Coagulation factor x formulations

Abstract

The invention relates to a formulation of a Factor X, comprising a non-ionic surfactant, a stabilizer, and a buffering agent, the formulation having a pH of 7-9. The invention further relates to said formulation for use as a medicament.

Description

[0001] P138550PC00

[0002] Title: Coagulation Factor X formulations

[0003] FIELD The invention relates to formulations comprising concentrations of more than 5 mg / mL of a Factor X, and to its use as a medicament, in particular for administration by injection.

[0004] BACKGROUND OF THE INVENTION

[0005] Coagulation Factor X is a vitamin K-dependent, liver-produced serine protease that serves in the coagulation cascade to form fibrin. Activation of FX occurs via proteolysis to release a small N-terminal 52 amino acid- activation peptide. The activated FX that is generated, FXa, is an active serine protease and has high homology to the trypsin family of serine proteases. Factor Xa converts prothrombin to thrombin in the presence of Factor Va, calcium and phospholipid during blood clotting (Versteeg et al., 2013. Physiol Review 93: 327–358).

[0006] A human coagulation factor X is indicated in adults and children with a rare, hereditary Factor X deficiency (about 1 in 1 million people) for routine prophylaxis to reduce the frequency of bleeding episodes, on-demand treatment and control of bleeding episodes, and perioperative management of bleeding. In addition, it may be used to reverse acquired coagulation factor deficiency induced by Vitamin K antagonist therapy in adult patients with a need for an urgent surgery / invasive procedure.

[0007] Presently, two commercially available FX concentrates are available for hereditary Factor X deficiency: Factor X P Behring, manufactured by CSL Behring, and Coagadex produced by Bio Products Laboratory. Reconstituted Factor X P Behring contains nominally 30-60 International Units (IU) / mL of native human coagulation FX and 30 IU / mL of human coagulation FIX per vial. Coagadex contains 100 IU / mL of native human coagulation FX. Both FX concentrates are administered by infusion at a rate not to exceed 20 mL / min.

[0008] BRIEF DESCRIPTION OF THE INVENTION

[0009] The invention provides a formulation of a Factor X, comprising more than 5 mg / mL, such as 5-20 mg / mL, of a Factor X, 0.05-5 mg / mL of a non-ionic surfactant, 5-150 mg / mL of a stabilizer, and 0.5-10 mg / mL of a buffering agent, theformulation having a pH of 7-9, wherein the Factor X is a pro-enzyme or zymogen, a non-activated form of Factor X.

[0010] In embodiments, said formulation comprises about 10 mg / mL of a Factor X. In embodiments, said Factor X, after activation, has a specific activity of 1-100 IU / mg, such as 1-50 IU / mg.

[0011] In embodiments, said stabilizer is a non-metal salt, a sugar, a sugar alcohol, and / or an amino acid, preferably sorbitol.

[0012] In embodiments, said non-ionic surfactant is or comprises a polysorbate, such as a polyoxyethylene (20) sorbitan monooleate. A formulation of the invention preferably comprises 0.1 mg / mL of a non-ionic surfactant such as polyoxyethylene (20) sorbitan monooleate. In embodiments, a formulation of the invention comprises 50 mg / mL of a stabilizer such as sorbitol.

[0013] In embodiments, a formulation of the invention comprises a buffering agent which is or comprises L-arginine and L-glutamic acid. In embodiments, said buffering agent is 1.72 mg / mL L-arginine and 1.45 mg / mL L-glutamic acid. Of note, the buffering capacity of said formulation may not be optimal as one of the pKa’s of arginine is 9.0 which is a full pH unit above the intended target pH of the formulation. However, surprisingly, such formulation of the invention was found to be stable even at elevated temperatures such as 5 °C and at room temperature, without aggregation of Factor X.

[0014] In embodiments, a formulation of the invention comprises a recombinant Factor X. Said Factor X, especially said recombinant Factor X, is preferably obtained from a eukaryotic cell-based expression system. Said Factor X, especially said recombinant Factor X, preferably is VMX-C001.

[0015] The invention further provides a lyophilized composition that provides a formulation according to the invention after reconstitution. Surprisingly, a formulation of the invention was found to be suitable for lyophilization, besides being stable at elevated temperatures such as 5 °C and at room temperature.

[0016] The invention further provides a formulation according to the invention, for use as a medicament. In embodiments, said medicament comprises 150 mg of Factor X. In embodiments, said medicament comprises 15 mL of the formulation according to the invention. In embodiments, said medicament is administered by injection, preferably by intravenous injection. In embodiments, said medicament is administered by an intravenous slow push injection. In embodiments, saidmedicament is administered by an intravenous injection, for example by a 10 seconds intravenous slow push injection. Surprisingly, a formulation of the invention was found to be suitable for injection, and allowed injection of a required amount of Factor X within a short time frame, such as within 1 minute or less, for example within 10 seconds. In contrast, state of the art formulations comprising Factor X such as Coagadex are to be provided by infusion.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1. Fluorescent intensity versus temperature plots for VMX-C001 at pH 5.5, 5% sucrose, and 0.01% PS80.

[0019] Figure 2. Bivariate fit of VMX-C001 Tagg by pH, salt, sugar, and surfactant. Note, each dot represents a single formulation.

[0020] Figure 3. Contour plots of pH interactions using JMP 12 software.

[0021] Figure 4. Graphical representation of SE-UPLC stability of initial VMX-C001 twelve week liquid stability following 25°C / 60%RH incubation, as well as freeze / thaw and shaking conditions. A: stability at 25°C / 60%RH, t=4 weeks. B: stability following freeze / thaw stress and shaking at 2-8°C. C: % Stability (isoaspartate quantitation) at 25°C / 60%RH, t=4 weeks. D: Stability (isoaspartate quantitation) at 2-8°C / 60%RH, t=12 weeks.

[0022] Figure 5. Graphical representation of SE-UPLC stability of initial VMX-C001 twelve week liquid stability following -80°C and 2-8°C incubation. A: stability at -80 °C, t=12 weeks. B: at 2-8°C, t=12 weeks.

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] Definitions

[0025] As are used herein, the singular forms “a”, “an” and “the”, are intended to include the plural forms as well.

[0026] As is used herein, the term “or” includes any and all combinations of one or more of the associated listed items, unless the context clearly indicates otherwise (e.g., if an “either....or” construction is used).

[0027] As are used herein, the terms “comprise” and “comprising”, and conjugations thereof, are open language and specify the presence of stated features but do not preclude the presence or addition of one or more other features.It will be understood that when a particular step of a method is referred to as subsequent to another step, it can directly follow said other step or one or more intermediate steps maybe carried out before carrying out the particular step, unless specified otherwise.

[0028] The term “recombinant”, as used herein, refers to a protein that is produced using recombinant DNA techniques known to the person skilled in the art. A recombinant protein may not be identical to its native counterpart, for example due to minor differences in the amino acid composition and / or differences in posttranslational modification such as glycosylation.

[0029] The term “native”, as used herein, refers to the endogenous form of a protein, polypeptide or nucleic acid as it naturally occurs in an animal, preferably in a mammal, more preferably in a primate, more preferably in a human. An example of a native protein is plasma-derived Factor X (pd-FX), which corresponds to the naturally occurring human coagulation Factor X isolated from plasma.

[0030] As used herein, the suffix ‘a’, in the context of a coagulation factor, denotes the activated form of a coagulation factor, whereas the absence of the suffix indicates the non-activated (zymogen) form.

[0031] The term “mature coagulation FX”, as used herein, refers to an non-activated coagulation FX protein that is composed of a light and a heavy chain that are linked by a disulfide bond. This FX protein is also referred to as proprotein FX, or zymogen FX. Activation of mature FX is mediated by binding to, for example, a FIXa: FVIIIa complex (intrinsic pathway) or tissue Factor-FVII complex (extrinsic pathway), resulting in proteolytic cleavage of the activation peptide from the heavy chain. As is used herein, a mature coagulation FX comprises within its sequence the polypeptide region that resembled activated coagulation Factor Xa (FXa) upon proteolytic activation by cleavage of the activation peptide.

[0032] The term “FXa DOAC”, as used herein, refers to a direct FXa anticoagulant or inhibitor, for example an oral direct FXa inhibitor. FXa DOACs are small compound inhibitors that bind to and reduce, or even halt, the activity of coagulation FXa. The group of FXa DOACs includes, but is not limited to, rivaroxaban, apixaban, edoxaban, betrixaban, darexaban, otamixaban, eribaxaban, letaxaban, LY517717, and aXInd 813893.

[0033] The term “pharmaceutical composition”, in the context of the invention, refers to a combination of a protein of the invention with a pharmaceutically acceptablecarrier, inert or active, making the composition suitable for therapeutic use in vivo or ex vivo. Said composition may be, for example, an aqueous composition or a lyophilized composition.

[0034] The term “pharmaceutically acceptable”, as used herein, refers to a nontoxic material that is compatible with the physical and chemical characteristics of a protein of the invention and does not interfere with the effectiveness of the biological activity of said protein.

[0035] The invention is directed to a composition comprising at least 5 mg / mL, preferably 5-20 mg / mL, preferably about 10 mg / mL of a coagulation Factor X protein, 0.05-5 mg / mL of a non-ionic surfactant, 5-150 mg / mL of a stabilizer, and 0.5-10 mg / mL of a buffering agent, the formulation having a pH of 7-9. Said composition can be stored for at least 6 months at 5 °C or less, and has acceptable viscosity, turbidity, visible and subvisible particle counts.

[0036] Human coagulation factor X (FX; EC 3.4.21.6 according to IUBMB Enzyme nomenclature) is a glycoprotein that circulates as an non- activated zymogen (proenzyme). Upon proteolytic activation, the activated form termed Factor Xa (FXa) functions as a serine protease enzyme that selectively cleaves Arg-Thr and then Arg-Ile bonds in prothrombin to form thrombin. Other name(s) for FX are fibrinogenase; thrombase; Stuart factor; Stuart-Prower factor; thrombin-C; E thrombin; B-thrombin; and Y-thrombin. FX is produced as a preproprotein that is converted to a mature two-chain form by a subtilisin-like protease termed furin. Mature FX is a glycoprotein composed of a heavy chain and a light chain, which are held together by a disulfide bond. Mature FX is activated by proteolytic release of the N-terminal activation peptide of the heavy chain by either FVIIa / tissue factor or by FIXa / FVIIIa. Activated FXa converts prothrombin to thrombin in the presence of factor Va, Ca2+ and phospholipids.

[0037] The amino acid sequences of FX and FXa are known. Reference is made to entry P00742 in the UniProt database. The gene encoding FX is known as entry GC13P113122 in GeneCards, entry 3528 in the HGNC database, entry 2159 in NCBI, and entry ENSG00000126218 in the Ensembl database. An amino acid sequence of human FX preproprotein is provided herein below as SEQ ID NO:1.

[0038] Factor X protein variants comprising an insertion in a BLTX 99-loop render said variants less susceptible for inhibition by a direct FXa inhibitor (or FXaDOAC) (Verhoef et al., 2017. Nature Comm 8: 528). Moreover, or in addition, FX variants comprising a substitution of phenylalanine 174 (F174 of FXa; F396 of human coagulation Factor X), for example for alanine (F396A), isoleucine (F396I), or serine (F396S), are able to bypass a direct oral FXa inhibitor (FXa DOAC) (Schreuder et al., 2024. J Thrombosis Haemostasis 22: 2211-2226). These variants counteract the effects of 3 widely used FX anticoagulants, apixaban, edoxaban, and rivaroxaban. Moreover, further FX variants comprising a S395 substitution of human coagulation Factor X or comprising a combination of F396 and S395 substitutions of human coagulation Factor X may be formulated in a formulation according to the invention.

[0039] In embodiments, said coagulation Factor X protein is a non- active recombinant FX that is activated by proteolytic cleavage once administered to a patient. In embodiments, said recombinant Factor X protein is a variant that is less sensitive to FXa DOAC. Said variant can be produced using recombinant DNA techniques known to the person skilled in the art.

[0040] In embodiments, the formulation according to the invention comprises recombinant, non-activated Factor X, including a recombinant variant that is less sensitive to FXa DOAC inhibition. The administered product is not activated Factor X and does not comprise substantial amounts of activated Factor X at the time of administration, such as not more than 0.1%, preferably not more than 0.01% of total FX.

[0041] A Factor X protein or variant thereof (FX) preferably is 70-100% identical to the amino acid sequences of the heavy and light chains of VMX-C001 as provided herein as SEQ ID NO:2 and SEQ ID NO:3, such as at least 80% identical, at least 90% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical to the amino acid sequence of SEQ ID NOs 2 and 3 over their full lengths.

[0042] The formulation according to the invention comprises Factor X in its nonactivated form, which is activatable in vitro or in vivo by proteolytic cleavage of the activation peptide at the N-terminus of the heavy chain, mediated by the Factor VHa / tissue factor complex, and / or by the Factor IXa / F actor Villa complex. Once activated, the specific activity of activated Factor X maybe 1-100 lU / mg, including 1-50 lU / mg, such as 2-20 lU / mg, 5-10 lU / mg, for example 5 lU / mg, 6 lU / mg, 7 lU / mg, 8 lU / mg, 9 lU / mg or 10 lU / mg and all values in between.The specific activity of an activated Factor X such as activated VMX-C001 may be determined by, for example, a coagulation activity assay for tissue factor-mediated thrombin generation (Stief et al., 2008. Clin Applied Thrombosis / Hemostasis 14: 303-318) by employing, for example, Factor X deficient plasma. Said Factor X deficient plasma may be generated by affinity immunoadsorption using antibodies directed towards FX. In brief, thrombin formation in plasma or in Factor X deficient plasma may be triggered by addition of tissue factor (TF) and phospholipids. TF indirectly activates a Factor X, such as VMX-C001. Thrombin generation may be continuously monitored by means of a thrombin-specific chromogenic or fluorogenic substrate. The amount of thrombin activity is determined in comparison with a thrombin standard, resulting in a so-called thrombogram (Tripodi, 2016. Clin Chem 62: 699-707). Commercially available assays for determining the specific activity of a FX following activation include, for example, Technothrombin (Technoclone), Thrombinoscope (Stago), and Innovance ETP (Siemens Healthcare).

[0043] The activity of an activated Factor X such as activated VMX-C001 may be determined by determining prothrombin time (PT) and / or an activated partial thromboplastin time (aPTT), as is known to a person skilled in the art. In such assays, Factor X is activated to Factor Xa by TF-Factor VIIa complex (PT) or by the intrinsic tenase complex (aPTT), and activated Factor Xa promotes thrombin formation and clot generation. Examples of automated instruments for such analyses include the CS-2500 and CS-5100 systems (Siemens Healthcare and the cobas® t 511 coagulation analyzer (Roche Diagnostics).

[0044] Separate therefrom, or in addition thereto, the activity of an activated Factor X such as activated VMX-C001 may be determined with a synthetic factor Xa substrate that has a chromophore attached to one end. In such assays, Factor X is first converted to activated Factor Xa within the assay system, whereafter the substrate is cleaved by factor Xa, resulting in a detectable colorimetric or fluorometric change, which can be quantified. Extent of color change is directly proportional to enzyme activity. Commercial Fx assays include the Factor Xa Activity Fluorometric Assay Kit (MilliporeSigma), Verachrom VXS-25 (Provision Kinetics, Inc., Arlington, WI), Chromogenix S-2222 and Chromogenix S-2765 (DiaPharma Inc., West Chester, OH).The specific activity of a Factor X such as VMX-C001, following activation under assay conditions, maybe 1-100 IU / mg, such as 1-50 IU / mg, including 2-20 lU / mg, 5-10 lU / mg, for example 5 lU / mg, 6 lU / mg, 7 lU / mg, 8 lU / mg, 9 lU / mg and 10 lU / mg and all values in between.

[0045] Compositions

[0046] The compositions described herein are compositions comprising non-activated Factor X (FX) unless explicitly stated otherwise. References to “FXa” in this section refer to the activated form and are not intended to describe the administered product in these compositions.

[0047] An aqueous composition according to the invention may comprise 5-20 mg / mL of non-activated FX, such as at least 5 mg / mL, at least 6 mg / mL, at least 7 mg / mL, at least 8 mg / mL, at least 9 mg / mL, at least 10 mg / mL, at least 11 mg / mL, at least 12 mg / mL, at least 13 mg / mL, at least 14 mg / mL, at least 15 mg / mL, at least 16 mg / mL, at least 17 mg / mL, at least 18 mg / mL or at least 19 mg / mL. A composition of the invention may comprise 5-15 mg / mL, such as 8-12 mg / mL, such as about 9 mg / mL, about 10 mg / mL, or about 11 mg / mL FX.

[0048] A composition according to the invention provides an aqueous formulation of a sufficient dose of FX protein, allowing injection of the required dose to an individual in need thereof. Remarkable, said formulation is stable at 4 °C for at least 6 months, thereby providing a ready-to-use formulation that does not need to be reconstituted. Hence, a formulation according to the invention provides a convenient, ready-to-use antidote to FXa DOAC for use in emergency situations such as urgent surgical procedures that require a quick reversal of anticoagulation.

[0049] In embodiments, said FX is a recombinant FX that is obtained from a cellbased expression system. Suitable cell-based expression systems are known in the art and include mammalian expression systems such as HeLa cells, Human Embryonic Kidney (HEK) 293 cells, Hep G2 cells, Chinese Hamster Ovary (CHO) cells and Baby hamster kidney (BHK) cells. In embodiments, a recombinant FX such as VMX-C001 is produced in a CHO cell line, such as a CHO cell line lacking dihydrofolate reductase (DHFR) activity. CHO cell lines and derivatives thereof can be obtained from a number of biological resource centers such as the European Collection of Authenticated Cell Cultures (ECACC). Expression of a vitamin K-dependent enzyme such as FX in mammalian cells may benefit from co-expressionof a vitamin K-dependent epoxide reductase (VKOR), a γ-glutamyl carboxylase, and / or of a protease such as furin, a paired basic amino acid cleaving enzyme that is a member of the subtilisindike proprotein convertase family, as has been described for the related coagulation Factor IX protein (Liu et al., 2014. Protein J 33: 174-183).

[0050] An aqueous composition according to the invention has a pH between 6.5 and 8.5, including endpoints, such as 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, and 8.5, preferably at a pH of about 7.6. Said composition is maintained at the indicated pH by the presence of a buffering system. Said buffering system, also termed buffer, may not only function to regulate shifts in pH, but also to stabilize proteins. In embodiments, said buffer may be selected, for example, from an amino acid such as histidine, aspartic acid, arginine, lysine, and / or glutamic acid, sodium bicarbonate, sodium bicarbonate / sodium carbonate, 4-(2-hydroxyethyl)piperazine-l-ethanesulfonic acid, N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-{[l,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino}ethane-l-sulfonic acid (TES), 3-N-bis-(hydroxyethyl)-amino-2-hydroxypropane sulphonic acid (DIPSO), MOBS (4-(N-morpholino)butanesulfonic acid) (MOBS), 3-N-tris-(hydroxymethyl)-methylamino-2-hydroxypropanesulphonic acid sodium salt (TAPSO). Phosphoric acid is preferably not included as a buffer.

[0051] In embodiments, said buffer is selected from one or more amino acids such as histidine, aspartic acid, arginine, lysine, and / or glutamic acid, such as Glu-Arg, Asp-Arg, and Glu-Lys. In embodiments, the buffer comprises L-arginine and L-glutamic acid. In embodiments, the buffer consists of the amino acids L-arginine (Arg) and L-glutamic acid (Glu).

[0052] Although Arg-Glu have been reported to suppress mAb aggregation at weakly acidic to neutral pH (Kheddo et al., 2014. Int J Pharm 473: 126-133), it was found that Arg-Glu suppresses FX aggregation at slightly basic conditions between pH 6.5 and pH 8.5, such as 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, and 8.5, preferably at a pH of about 7.6.

[0053] In addition, Arg-Glu concentrations of more than 200 mM in the final formulation maybe necessary for optimal protein solubility (Kheddo et al., 2014. Int J Pharm 473: 126-133). It was surprisingly found that Arg-Glu concentrations of below 100 mM, and even below 50 mM, function well in stabilizing FX proteinssuch as VMX-C001 and prevent their aggregation and precipitation. Suitable concentrations of Arg-Glu are between 1 mM and 50 mM, such as between 5 mM and 20 mM, including 2 mM, 5 mM, 10 mM, 15 mM, 20 mM, 30 mM, and 40 mM. Arg-Glu concentrations are preferably equimolar, such that each of Arg-Glu is present in a Factor X formulation of the invention at a concentration of between 1 mM and 50 mM, such as between 5 mM and 20 mM, including 2 mM, 5 mM, 10 mM, 15 mM, 20 mM, 30 mM, and 40 mM. Suitable concentrations of Arg-Glu are 5-15 mM, especially 10 mM.

[0054] In embodiments, the composition comprises L-arginine and L-glutamic acid at equimolar concentrations (for example about 10 mM each) to provide buffering and contribute to suppression of aggregation at about pH 7.6.

[0055] In embodiments, a formulation of the invention comprises 1-1000 mM of a stabilizer to prevent the formation of aggregates and particles. In embodiments, said stabilizer is a sugar or a sugar alcohol. A sugar includes, for example, a disaccharide sugar such as sucrose, trehalose, maltose, and lactose. A sugar alcohol includes, for example, glycerol, erythritol, threitol, xylitol, mannitol and sorbitol. Said sugar or sugar alcohol may act as a stabilizing agent, for example by increasing the free energy of unfolding of a protein, thereby thermodynamically favoring the native folded state.

[0056] In embodiments, said stabilizer is a sugar alcohol such as sorbitol or mannitol. In embodiments, said stabilizer is sorbitol. Said sugar alcohol such as sorbitol or mannitol is present in a formulation of the invention at a concentration of between 0.05 M and 1 M, such as between 0.1 M and 0.5 M, including between 0.2 M and 0.4 M such as 0.27 M (5 % (m / v)). As is shown in the examples, about 0.27 M (5 % (m / v)) of a sugar alcohol such as sorbitol showed the highest main peak retention of FX, in the absence of glycine and mannitol.

[0057] As is shown in the examples, stability data distinguish sorbitol from sucrose with respect to turbidity development at 2-8°C, wherein turbidity increases slightly more in sucrose -containing formulations than in sorbitol-containing formulations, indicating improved physical stability in the presence of sorbitol.

[0058] Furthermore, formulations comprising a combination of mannitol and sucrose show an increased formation of both high molecular weight (HMW) FX species and low molecular weight (LMW) FX species, suggesting enhanced aggregation and fragmentation of FX under storage conditions. In contrast, formulations comprisingsorbitol as stabilizer demonstrate reduced levels of such degradation-related FX species, thereby supporting improved structural integrity of the Factor X polypeptide.

[0059] In addition, chemical stability analysis indicates that isoaspartate formation is lower in sorbitol-containing formulations compared to formulations comprising sucrose alone or in combination with mannitol. Reduced isoaspartate levels reflect decreased deamidation and improved preservation of protein primary structure.

[0060] In embodiments, a formulation of the invention comprises 0.01-5 mg / mL of a further stabilizer such as a non-ionic surfactant. A non-ionic detergent comprises molecules with uncharged head groups. A non-ionic detergent is considered to be non- denaturing and useful in formulations to preserve the structure of protein such as FX. In addition, a non-ionic detergent may prevent protein adsorption to hydrophobic surfaces and prevent the formation of protein aggregates and particles, thereby acting as a stabilizer. Examples of such further stabilizer are polyoxyethylene (23) lauryl ether, N-octanoyl-N-methylglucamine, saponin, a sorbitan ester, and an ethylene oxide / propylene oxide block copolymer such as Kolliphor® P407.

[0061] A preferred non-ionic detergent as a further stabilizer is a sorbitan ester such as an ethoxylated sorbitan ester, also known as polysorbate or TWEEN®. They are an important class of emulsifiers used in a variety of settings, including pharmaceuticals and food. A preferred further stabilizer is polysorbate 80 or polyoxyethylene (20) sorbitan monooleate ((2R)-2-[(2R,3R,4S)-3,4-dihydroxyoxolan-2-yl]-2-hydroxyethyl (9Z)-octadec-9-enoate), whereby the number 20 refers to the total number of oxyethylene (–CH2CH2O–) groups that are present in the molecule. Said polysorbate 80 is commercially available as TWEEN® 80 (Croda Americas Ltd) or Span® 80 (Croda International PLC).

[0062] A formulation of the invention may comprise 0.02- 1 mg / mL of a further stabilizer, such as between 0.05 and 0.5 mg / mL, between 0.08 and 0.12 mg / mL, between 0.09 and 0.11 mg / mL, including about 0.1 mg / mL of a further stabilizer such as polysorbate 80.

[0063] In embodiments, the invention provides a formulation comprising about 5-20 mg / mL of a Factor X such as VMX-C001, 0.05-5 mg / mL of a non-ionic surfactant, 5-150 mg / mL of a stabilizer, and 0.5-10 mg / mL of a buffering agent, the formulation having a pH of 7-9.A formulation of the invention comprises no visible particles. The number of particles having a size of >10 µm is less than 6000 per container. The number particles >25 µm is less than 600 per container. Preferably, these particulate limits are maintained after prolonged storage at elevated temperatures, after repeated freeze-thaw cycles, and / or after exposure to mechanical stress, including shear stress.

[0064] With visible particles is meant that particle(s) can be observed with the naked eye by a person skilled in the art, optionally using a means for magnification, such as a magnifying glass. For regulatory purposes, there is a distinction between visible and non-visible particulate matter. A visible particulate is defined as any particulate that can be detect-ed with an unaided eye. Typically, visible objects are defined as objects that are 0.05 mm or larger. With the term “visible particles”, as used in the present invention, is meant particles that are 0.05 mm or larger, preferably 0.1 mm or larger, more preferably 0.2 mm or larger, more preferably 0.5 mm or larger, most preferably 1 mm or larger.

[0065] Micron-sized protein aggregates and particles (subvisible particles, SVP) are important quality attributes of therapeutic protein formulations due to their risk of enhancing an immunogenic response. A quantification of SVP larger than 10 pm and of SVP larger than 25 pm is required. Currently, the acceptance criteria for SVP are: not more than (NMT) 6000 SVP >10 µm per container and NMT 600 SVP >25 µm per container. Subvisible particles (SVP) < 10 µm have to be monitored, however specific acceptance criteria are not defined. The compendial methods Ph. Eur. 2.9.19 and USP 787 / 788 specify light obscuration as method for subvisible particle counts, such as dynamic light scattering and static light scattering.

[0066] Further suitable methods include nanoparticle tracking analysis, light microscopy, electrical sensing zone, flow-imaging technology, resonant mass measurement, electron microscopy, Fourier transform infrared microscopy, and Raman microscopy.

[0067] Methods for determining particle formation are known to a person skilled in the art and include light obscuration, by which the size of a particle in a product is determined by the amount of light that it blocks; electrical sensing zone (Coulter), by which the size of a particle is measured in terms of the change in resistance as the particle passes through a microchannel, flow imaging microscopy, by which a high-speed camera records images as a sample flows through a flow cell, therebycapturing images of particles in real-time, or laser diffraction, by which the size of a particle is determined by measuring the angle of the scattered light.

[0068] In addition, a formulation according to the invention should have acceptable viscosity, turbidity, osmolality and injectability.

[0069] The viscosity of a composition may be a limiting factor when administering a pharmaceutical composition comprising a Factor X such as VMX-C001. Especially when administration it by injection, which may require a fast injection rate for injecting a volume of, for example, 2-20 mL. Viscosity (centipoise (cP) or mPa*s) may be determined by a rotational viscometer such as a HAAKE MARS II rheometer (Thermo Fisher Scientific) at a shear rate of 200 / s at 20 °C, or by Cone-Plate Rheometry, for example by using a RSX Cone Plate Rheometer (AMETEK Brookfield) at 1000 s-1.

[0070] A viscosity of less than 20 cP is considered feasible, while a viscosity of less than 15 cP may be desired for an injectable composition. A formulation according to the invention comprising up to 20 mg / mL of a Factor X such as VMX-C001, 0.05-5 mg / mL of a non-ionic surfactant, 5-150 mg / mL of a stabilizer, and 0.5-10 mg / mL of a buffering agent, the formulation having a pH of 7-9, was found to have a viscosity of 1,5 cP or less, such as 1.2- 1.4 cP.

[0071] Turbidity or opalescence is a measure of the relative clarity of a formulation by determining the scattering effect that particles may have on light. Turbidity may be determined by any method known in the art, including visual evaluation and dynamic light scattering (DLS) / static light scattering (SLS) by determining the difference in intensity between the transmitted beam and the incident beam.

[0072] Suitable instruments include DynaPro® NanoStar® (Wyatt Technology Corp, Santa Barbara, CA) and NEPHELOstar Plus (BMG Lab tech). Turbidity is preferably determined according to standards such as USP <855>, Ph. Eur. 2.2.19, USEPA Method 180.1, or ISO7027.

[0073] There are two standard units for reporting turbidity: Formazin Nephelometric Units (FNU) from ISO 7027 and Nephelometric Turbidity Units (NTU) from USEPA Method 180.1.

[0074] A turbidity of less than 20 NTU is desired for an injectable composition. As is shown in Figure 2, a formulation according to the invention comprising up to 20 mg / mL of a Factor X such as VMX-C001 was found to have a turbidity of less than 20 NTU.A formulation comprising a Factor X such as VMX-C001 according to the invention may cause tonicity-related discomfort / pain upon injection such as subcutaneous injection, which may be associated with the osmolality of the composition. Osmotic pressure of an injectable composition can be expressed as either osmolality or osmolarity. Osmolality is defined as the number of milliosmoles of solute per kilogram of solvent and can be calculated experimentally using sodium chloride equivalents or determined with an osmometer, as is known to a person skilled in the art. Methods for determining the osmolality of a composition are known, including the use of a freezing point depression osmometer, a vapor pressure osmometer or a membrane osmometer. Suitable osmometers include a freezing point depression osmometer such as an Osmomat 3000 D-M (Gonotec GmbH) and Precision Systems Osmette II™ and Osmette III™ Osmometers (Thermo Fisher Scientific). The upper osmolality limit of a formulation for subcutaneous injection in general is below 1000 mOsm / kg for small-volume injections (<100 mL) and below 500 mOsm / kg for large-volume injections (>100 mL). Several options are available for minimization of hypertonicity-induced pain upon product administration. As is shown in the examples, a formulation comprising a Factor X such as VMX-C001 according to the invention has an osmolality of 300-310 mOsm / kg.

[0075] As an alternative, a formulation of the invention may be provided as a lyophilized formulation which, upon reconstitution, provides a formulation of the invention comprising 5-20 mg / mL of a Factor X such as VMX-C001, 0.05-5 mg / mL of a non-ionic surfactant, 5-150 mg / mL of a stabilizer, and 0.5-10 mg / mL of a buffering agent and which is buffered to a pH of about 7.6.

[0076] Methods for lyophilizing a formulation of the invention comprising 5-20 mg / mL of a Factor X such as VMX-C001, 0.05-5 mg / mL of a non-ionic surfactant, 5-150 mg / mL of a stabilizer, and 0.5-10 mg / mL of a buffering agent and which is buffered to a pH of about 7.6, are known to a person skilled in the art. For example, lyophilization, also termed freeze drying or cryodesiccation, is a dehydration process that involves freezing the product and lowering pressure, for example by applying a vacuum, to thereby dry the product by removing water firstly by sublimation, followed by desorption.

[0077] In embodiments, the formulation of the invention is stored in a frozen state, for example at approximately -80°C or -20°C, for a period of time, for example forat least 3 months, at least 6 months, at least 1 year, at least 1.5 years, or at least 2 years.

[0078] In embodiments, for example after storage in a frozen state, the formulation of the invention may be stored below room temperature, for example at approximately 4°C, for a period of at least 3 months, at least 6 months, at least 1 year, at least 1.5 years, or at least 2 years.

[0079] Methods of use

[0080] A formulation comprising about 5-20 mg / mL of a Factor X such as VMX-C001, 0.05-5 mg / mL of a non-ionic surfactant, 5-150 mg / mL of a stabilizer, and 0.5-10 mg / mL of a buffering agent may be used for intravenous (i.v.) administration of a Factor X such as VMX-C001, including i.v. injection and i.v. infusion. Said formulation comprising about 5-20 mg / mL of a Factor X such as VMX-C001, 0.05-5 mg / mL of a non-ionic surfactant, 5-150 mg / mL of a stabilizer, and 0.5-10 mg / mL of a buffering agent is for use in a method of restoring blood coagulation in patients taking a Factor Xa Direct Oral Anticoagulant (FXa-DOAC). For example, said formulation may stop severe bleeding and eliminate bleeding risk during surgery in patients taking a FXa DOAC, including emergency or urgent surgical procedures, for example in patients who have sustained serious trauma or significant blood loss where rapid reversal of anticoagulation is required.

[0081] Said formulation comprising 5-20 mg / mL of a Factor X such as VMX-C001, 0.05-5 mg / mL of a non-ionic surfactant, 5-150 mg / mL of a stabilizer, and 0.5-10 mg / mL of a buffering agent may be directly injected, for example as a single injection. Said formulation may be injected at a single dosage within a short period of time, such as within 2 minutes, within 1 minute, within 30 seconds, within 20 seconds, or even within 10 seconds, to a person in need thereof, for example to restore blood coagulation in a patient taking a FXa-DOAC, or to restore or support blood coagulation in a person suffering from a genetic coagulation disorder, such as an inherited Factor X deficiency, including a pediatric patient, for example a neonate, infant, child or adolescent up to and including 18 years of age.

[0082] In embodiments, the formulation is administered no later than 24 hours, no later than 20 hours, no later 15 hours, no later than 10 hours, no later than 5 hours, or immediately following the patient’s last dose of a FXa-DOAC.

[0083] In embodiments, the formulation is administered as soon as possible after documentation of a plasma factor Xa direct oral anticoagulant (FXa-DOAC) levelmeeting or exceeding a predefined threshold. For example, administration may occur within 5 hours, within 2 hours, or immediately following determination that the plasma FXa-DOAC level is >75 ng / mL, or >0.85 lU / mL as determined using a heparin -calibrated anti-FXa assay, wherein the value expressed in lU / mL is obtained by comparison of residual Factor Xa activity in the sample to a calibration curve generated using heparin standards of known International Unit concentration.

[0084] In embodiments, the formulation is administered as a slow intravenous push injection over a short period of time, for example about 5-30 seconds, including approximately 25 seconds, approximately 20 seconds, approximately 15 seconds, approximately 10 seconds, and approximately 5 seconds. Said formulation may be administered via intravenous injection instead of intravenous infusion, as the formulation according to the invention comprises Factor X in its non-activated form, which is activated in vivo after administration. Because of its non-activated form, the formulation may be administered at a higher dose than a corresponding activated Factor Xa formulation, thereby enabling bolus injection rather than continuous infusion. In vivo, the administered non-activated FX is converted to activated FXa by proteolytic activation, including proteolytic activation by the tissue factor-FVIIa complex (extrinsic pathway) and / or by the FIXa-FVIIIa complex (intrinsic pathway). In contrast, activated forms of Factor X (FXa) due to their enzymatic activity, are not suitable for administration over a short period of time, such as within approximately 10 seconds by an intravenous push injection, as rapid administration may lead to uncontrolled or indiscriminate activation of coagulation.

[0085] A formulation according to the invention, comprising 5-20 mg / mL of a Factor X such as VMX-C001, may be provided to an individual in need thereof at a dose of 10-50 lU / kg, including 10-30 lU / kg. This means that 900-2700 IU are to be provided to an individual weighing 90 kg. For example, with an activity of 10 lU / mg, this totals to 90-270 mg of a Factor X such as VMX-C001. This means that a volume of between 4.5 mL and about 55 mL may be provided to an individual in need thereof for a formulation comprising 5-20 mg / mL of a Factor X such as VMX-C001. For example, a dose of 900-2700 lU / patient would correspond to a volume of 9 mL for the smallest dose, and of 27 mL for the largest dose, in case a formulation with a concentration of 10 mg / mL is used. For example, a dose of 150-200mg / patient, such as 150 mg / patient, 160 mg / patient, 170 mg / patient, 180 mg / patient, or 190 mg / patient, would correspond to a 15-20 mL injection in a short push directly from the syringe, in case a formulation with a concentration of 10 mg / mL is used.

[0086] In embodiments, the formulation is administered at a fixed dose of 150 mg per patient at a concentration of 10 mg / mL Factor X.

[0087] As an alternative, or in addition, a formulation according to the invention, comprising 5-20 mg / mL of a Factor X, may be provided to an individual in need thereof at a fixed dose of 100-250 mg, including 125-200 mg, including 150-175 mg, such as 150 mg, 160 mg or 170 mg per individual. In embodiments, a formulation of the invention comprising 5-20 mg / mL of a Factor X such as VMX-C001, 0.05-5 mg / mL of a non-ionic surfactant, 5-150 mg / mL of a stabilizer, and 0.5-10 mg / mL of a buffering agent and which is buffered to a pH of about 7.6, may be used for parenteral administration of a FX such as VMX-C001, in particular for subcutaneous or intravenous injection. In embodiments, the fixed-dose intravenous injection is used to rapidly restore coagulation in patients receiving an FXa-DOAC in circumstances where intravenous infusion may be impractical due to substantial blood loss or hemodynamic instability.

[0088] In embodiments, a formulation according to the invention, comprising about 5-20 mg / mL of a Factor X such as VMX-C001, 0.05-5 mg / mL of a non-ionic surfactant, 5-150 mg / mL of a stabilizer, and 0.5-10 mg / mL of a buffering agent is for use in a pediatric patient, including a neonate, an infant, a child and an adolescent up to and including 18 years of age. Said pediatric patient may suffer from a disorder requiring anticoagulant therapy and / or coagulation management, such as a genetic disorder. Such genetic disorder may include, for example, inherited Factor X deficiency or other congenital coagulation disorders requiring modulation of coagulation pathways, such as hemophilia A (FVIII-deficiency) and hemophilia B (FIX- deficiency). In such pediatric patient, the formulation may be administered to restore or support physiological coagulation balance.

[0089] In embodiments, a formulation according to the invention may be administered at a body weight-adjusted dose ranging from 1 mg / kg to 5 mg / kg, including 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, and 5 mg / kg. Such weight -based dosing is for example suitable for a pediatric patient, from birth up to and including 18 years of age, and allowsindividualized titration rather than administration of a fixed dose. For example, with a specific activity of 10 lU / mg, said body weight-adjusted dose may range 10 lU / kg to 50 lU / kg.

[0090] In embodiments, a formulation according to invention may be administered at a body weight- adjusted dose ranging from 1 mg / kg to 5 mg / kg, including 1 mg / kg, 1.5 mg / kg, 2 mg / kg, 2.5 mg / kg, 3 mg / kg, 3.5 mg / kg, 4 mg / kg, 4.5 mg / kg, and 5 mg / kg. Such weight-based dosing is for example suitable for a pediatric patient, from birth up to and including 18 years of age, and allows individualized titration rather than administration of a fixed dose.

[0091] In embodiments, a formulation according to the invention may be administered on demand or repeatedly, for example at regular time intervals. For example, the formulation may be administered to a pediatric patient every day, every other day, twice a week, once a week, once every two weeks, or once every month. Hence, a body weight-adjusted dose may be administered to a pediatric patient every day, every other day, twice a week, once a week, once every two weeks, or once every month.

[0092] In embodiments, a formulation according to the invention comprising 5-20 mg / mL of a Factor X such as VMX-C001, 0.05-5 mg / mL of a non-ionic surfactant, 5-150 mg / mL of a stabilizer, and 0.5-10 mg / mL of a buffering agent and which is buffered to a pH of about 7.6, may be used for parenteral administration of a FX such as VMX-C001, in particular for subcutaneous or intravenous injection. Said formulation maybe provided in a pre-filled syringe or a smart pen. Such parenteral administration, especially subcutaneous or intravenous administration, may be advantageous for treatment of a chronic disease, such as in the case of a FX deficiency. In embodiments, said parenteral administration, especially subcutaneous or intravenous administration, with a pre-filled syringe or a smart pen may be performed by an individual suffering from the chronic disease such as FX deficiency, hemophilia A or hemophilia B.

[0093] In embodiments, the invention provides a pharmaceutical composition comprising non-activated Factor X (FX), wherein the final drug product is supplied and administered as a fixed dose of 150 mg FX per patient at a concentration of 10 mg / mL, corresponding to a total injection volume of 15 mL. The composition is suitable for direct intravenous administration as a single slow intravenous pushinjection over approximately 10 seconds, wherein the administered FX is nonactivated at the time of administration and is activated in vivo by proteolytic cleavage. Once activated, the active Factor X has a specific activity of 1-100 lU / mg corresponding to 10-1000 lU / mL.

[0094] In embodiments, the composition comprises (i) 10 mg / mL non-activated Factor X, such as VMX-C001, (ii) 1.72 mg / mL L-arginine and 1.45 mg / mL L-glutamic acid as buffering agents, (iii) 50 mg / mL sorbitol as stabilizer, and (iv) 0.10 mg / mL polysorbate 80 as non-ionic surfactant.

[0095] Aspect 1

[0096] A composition comprising 5-20 mg / mL of a Factor X, such as VMX-C001.

[0097] Aspect 2

[0098] The composition according to aspect 1 comprising 10 mg / mL of a Factor X, such as VMX-C001.

[0099] Aspect 3

[0100] The composition of aspect 1 or aspect 2, wherein the Factor X, after activation, has a specific activity of 1-100 IU / mg, such as 1-50 IU / mg.

[0101] Aspect 4

[0102] The composition of any one of aspects 1-3, for use as a medicament.

[0103] Aspect 5

[0104] The composition for use of aspect 4, whereby the medicament comprises 150 mg of a Factor X, such as VMX-C001.

[0105] Aspect 6

[0106] The composition for use of aspect 4 or aspect 5, whereby the medicament comprises 15 mL of the composition.

[0107] Aspect 7

[0108] The composition for use according to any one of aspects 4-6, whereby the medicament is administered by injection.

[0109] Aspect 8

[0110] The composition for use of aspect 7, whereby the medicament is administered by intravenous injection.

[0111] Aspect 9The composition for use of aspect 7 or aspect 8, whereby the medicament is administered by intravenous slow push injection.

[0112] Aspect 10

[0113] The composition for use according to any one of aspects 7-9, whereby the medicament is administered by intravenous slow push injection over 10 seconds. Aspect 11

[0114] The composition according to any one of aspects 1-10, comprising

[0115] 0.05-5 mg / mL of a non-ionic surfactant,

[0116] 5-150 mg / mL of a stabilizer, and

[0117] 0.5-10 mg / mL of a buffering agent, the composition having a pH of 7-9.

[0118] Aspect 12

[0119] The composition of aspect 11, wherein the stabilizer is a non-metal salt, a sugar, a sugar alcohol, and / or an amino acid.

[0120] Aspect 13

[0121] The composition of aspect 11 or aspect 12, wherein the stabilizer is sorbitol.

[0122] Aspect 14

[0123] The composition of aspect 11, wherein the non-ionic surfactant is or comprises a polysorbate.

[0124] Aspect 15

[0125] The composition of aspect 11 or aspect 14, wherein the non-ionic surfactant is or comprises polyoxyethylene (20) sorbitan monooleate.

[0126] Aspect 16

[0127] The composition according to any one of aspects 11, 14 or 15, comprising 0.1 mg / mL of a non-ionic surfactant.

[0128] Aspect 17

[0129] The composition according to any one of aspects 11-13, comprising 50 mg / mL of a stabilizer.

[0130] Aspect 18

[0131] The composition of aspect 11, wherein the buffering agent is or comprises L-arginine and L- glutamic acid.

[0132] Aspect 19

[0133] The composition of aspect 11 or aspect 18, wherein the buffering agent is 1.72 mg / mL L- arginine and 1.45 mg / mL L-glutamic acid.Examples

[0134] Example 1

[0135] A high-throughput fluorescence-based protein stability assay, differential scanning fluorimetry that detects protein aggregation arising from thermally-induced protein denaturation was used to determine protein thermal stability in 72 different buffer conditions to monitor effects of pH, salt, sugar and surfactant type. Test samples were composed of protein, buffer, and dye (PROTEOSTAT, Enzo Life Sciences, p / n ENZ-51027). Changes in sample fluorescence were monitored in an Applied Biosystems 7300 PCR instrument over the temperature range of 25°C to 95°C in one-degree increments.

[0136] The fluorescent intensity versus temperature was plotted forming a sigmoid shaped curve expected for a two-state unfolding mechanism and the temperature at which the bulk of the protein becomes aggregated (Tagg) can readily be identified. An increase or decrease in Tagg can determine whether the protein is stabilized or destabilized relative to other conditions. The experimental matrix and data analysis is aided by design of experiment (DOE) software including ANOVA data evaluation to determine the significance of each excipient to the stability of the VMX-C001 protein.

[0137] A volume of 0.3 mL of 6.2 mg / mL VMX-C001 drug substance (Lot# DD-06530-002) was placed into pre-hydrated 10K MWCO dialysis cassettes (Thermo Scientific) and dialyzed at 2-8°C against 0.5 L 50 mM sodium citrate / 50 mM NaPO4 pH 4.5, 5.5, 6.5, and 7.5 buffers (2 changes of 0.5 L). The buffers were prepared using citric acid and monobasic sodium phosphate as the buffer components and 5N NaOH for pH adjustment, followed by a 0.2 pm polyethersulfone (Thermo Scientific) membrane filtration. Total dialysis time was three days. A noticeable precipitate was observed in the pH 4.5 dialyzed sample, thus this material was not processed any further. Post-dialysis, the three other samples were recovered, quantified at A280 using SoloVPE (CTech Repligen, Waltham, MA) and diluted to 1.8 mg / mL using the corresponding buffers. Each test solution was then added to a 96-well plate and subsequently spun-down to ensure that all liquid was at the bottom of the plate for consistent measurement prior to placing in the PCR instrument. The resulting fluorescent data was analyzed using JMP 13 software (JMP Statistical Discovery LLC, Cary, NC) to determine inflection point (Tagg) of a symmetric sigmoidal curve (Logistic 4p)A representative plot of the fluorescent intensity versus temperature at pH 5.5, 5% sucrose, and 0.01% PS80 is shown in Figure 1. Upon exposure to thermal stress, VMX-C001 material demonstrated less stability (a greater propensity to aggregate) at pH 5.5 as opposed to the higher pH conditions. A bivariate fit of the Tagg of the entire data set by pH, sodium chloride, sugar, and surfactant provides a view of relative trends as a function of each variable (Figure 2).

[0138] A standard least squares fitting of the data using the JMP 13 model indicates significance, with p < 0.0001 and an r-squared value of 0.97 (data not shown).

[0139] Looking at all possible effects and interactions indicates that pH significantly effects Tagg, sugar significantly effects Tagg, sodium chloride significantly effects (negatively) Tagg, and surfactant (especially PS80) significantly effects Tagg (data not shown). As pH provides the greatest effect to VMX-C001 stability, the contour plots of Figure 3 provide a visualization of the interactions of pH along with the other variables.

[0140] Combining DSF and HTS into a DOE matrix provides a quick and useful way of understanding the thermal stability of VMX-C001 as a function of formulation composition. Statistical evaluation of the VMX-C001 HTS DSF data using JMP 13 software indicates a number of significant effects, chief among them being sample pH with a maximum around pH 6.5. Using this information one can now proceed with additional formulation development focusing on this pH range as a means to achieve optimal VMX-C001 thermal stability.

[0141] Example 2

[0142] The high-throughput formulation evaluation indicated greater resistance to aggregation upon exposure to thermal stress around pH 6.5. The intention of this study was to aid in the selection of formulation conditions most suitable for the twelve-week stability study. An overview of the formulations is provided in Table 1. Samples were analyzed by the following methods:

[0143] -Visual Appearance

[0144] - Concentration by SoloVPE at A280 nm

[0145] - SE-UPLC

[0146] - pH.Table 1. VMX-C001 formulations

[0147] ID ID VMX- pH Glycine Sorbitol Mannitol Sucrose 0.01% # C001 (mM) PS80

[0148] (mg / mL)

[0149] 1 10 mM 5 6.2 50 1.6% 4% + 2 histidine 5 6.6 5% + 3 5 6.6 5% + 4 5 6.6 9% + 5 5 7 50 1.6% 4% + 6 10 mM 5 6.2 50 1.6% 4% + 7 arginine, 5 6.6 5% + 8 10 mM 5 6.6 5% + 9 glutamic 5 6.6 9% + 10 acid 5 7 50 1.6% 4% + 11 10 mM 7.2 50 1.6% 4% + 12 arginine, 7.6 50 1.6% 4% + 13 10 mM 7.2 5% + 14 glutamic 7.6 5% + 15 acid 8 50 1.6% 4% + 16 10 mM 8 50 1.6% 4% + arginine,

[0150] 10 mM

[0151] glutamic

[0152] acid,

[0153] 5mM

[0154] EDTA

[0155]

[0156] Formulation buffers were prepared according to Table 1. Briefly, all the buffer components were dissolved in Milli Q water, with the exception of PS80, and then followed by pH titration using 5N NaOH. All buffers were filtered through a 0.2 pm PES membrane before usage.

[0157] For Formulations 1-10, the 3.4 mg / mL VMX-C001 (Eot # DD-06897) material was placed into pre-hydrated 10K MWCO dialysis cassettes and dialyzed at 2-8°Cagainst IL of each buffer to be evaluated (3 changes of 1 liter). Total dialysis time of three days. The dialyzed material was subsequently concentrated by centrifugation to achieve a target concentration of >5 mg / mL. All test samples were then diluted with the appropriate buffers to a target concentration of 5 mg / mL VMX-C001. Polysorbate-80 was added to corresponding formulations to achieve a final concentration of 0.01% (w / v). Under aseptic conditions, the resulting VMX-C001 formulations were filtered through a 0.2 pm PVDF membrane and then 0.5 mL aliquots were dispensed into pre-sterilized depyrogenated borosilicate vials, stoppered and crimped with an aluminum seal.

[0158] For Formulations 11-16, 200 uL aliquots of 3.4 mg / mL VMX-C001 (Lot # DD-06897) material was placed into pre-hydrated 10K MWCO dialysis cassettes and dialyzed at 2-8°C against 100 mL of each buffer to be evaluated (3 changes of 100 mL). Total dialysis time of 24 hours. Polysorbate-80 was added to corresponding formulations to achieve a final concentration of 0.01% (w / v). Resulting samples were tested only by SE-UPLC.

[0159] All formulations 1-10 appeared as clear, colorless solutions (Table 6-2). All ten formulations had pH values much less than expected post dialysis potentially due to Donnan effects which are known to cause an offset in pH from the target value established with the diafiltration buffer during the concentration and diafiltration of charged proteins in which charged particles near a semi-permeable membrane fail to distribute evenly across the two sides of the membrane. To compensate for the pH offset caused by the Donnan effect, diafiltration buffers with pH and excipient values offset from the ultrafiltrate pool specifications can be used.

[0160] Example 3

[0161] Size exclusion chromatography (also known as gel filtration chromatography) separates the molecular forms of a protein based primarily on their hydrodynamic radius. In this method, larger molecular species (e.g. aggregates and oligomers) elute earlier than the desired monomeric species as pre-peaks. Smaller molecular species (e.g. degradation products and fragments) elute later as post-peaks. SE-UPLC was performed on the VMX-C001 stability samples using a Agilent AdvanceBio SEC 200A 1.9pm, 4.6x300 mm column and 10 mM phosphate, 300 mM arginine-HCl, pH 6.8 mobile phase on a Thermo Vanquish UHPLC system with Chromeleon processing software. The mobile phase was 10 mM phosphate, 300 mMarginine-HCI, pH 6.8. 10 pg sample was loaded by injection. Flow rate was 0.3 mL / min with isocratic elution.

[0162] SE-UPLC showed that extensive degradation at T=0 was observed with higher purities values found in the pH 7.0 samples. As with any coagulation protein, the binding of metals can affect both structure and activity, thus 5 mM EDTA was added to determine if chelating any metals that might be present would result in reducing the HMW species. Unfortunately, this was not the case (data not shown). Additional experimentation over the range of pH 7.0 -8.0 using an arginine / glutamic acid buffer resulted in purity levels near 89% in a pH 8.0 formulation containing glycine, mannitol and sucrose Table 2. Following six days of storage at 2-8°C a 3% decrease in purity of a formulation containing 5% sorbitol, pH 7.6, provided assurance that additional development should target formulations around this pH value.

[0163] Table 2. Tabular results of VMX-C001 pH 7-8 formulation screen.

[0164] SE-UPLC (% purity) Sample composition

[0165] HMW Main LMW 50 mM glycine, 1.6% mannitol,

[0166] 8.4 86.7 5.0 4% sucrose, 0.01% PS80, pH 7.2

[0167] 50 mM glycine, 1.6% mannitol,

[0168] 7.3 88.0 4.7 4% sucrose, 0.01% PS80, pH 7.6

[0169] 5% sorbitol, 0.01% PS80, pH 7.2 8.4 86.5 5.1 5% sorbitol, T=0 7.1 88.0 4.9 0.01% PS80,

[0170] 10 mM T=6 days (2- pH 7.6 9.2 85.0 5.8 arginine, 8°C)

[0171] 10 mM 50 mM

[0172] glutamic glycine, 1.6% Post dialysis 6.4 89.1 4.5 acid mannitol, 4%

[0173] sucrose,

[0174] 0.01% PS80, T=1 day (2-8°C) 6.7 88.0 5.3 pH 8.0

[0175] 5 mM EDTA, 50 mM Glycine,

[0176] 1.6% Mannitol, 4% Sucrose,

[0177] 6.4 88.8 4.8 0.01%

[0178]

[0179] PS80, pH 8.0

[0180] Additional experimentation over the range of pH 7.0 -8.0 using an arginine / glutamic acid buffer resulted in purity levels near 89% in a pH 8.0 formulation containing glycine, mannitol and sucrose Table 6-4. Following six daysof storage at 2-8°C, a 3% decrease in purity of a formulation containing 5% sorbitol, pH 7.6, provided assurance that additional development should target formulations around this pH value.

[0181] Example 4

[0182] Initial high-throughput formulation evaluation indicated greater resistance to aggregation upon exposure to thermal stress around pH 6.5. A subsequent study, examining buffer species over this target pH range, demonstrated that VMX-C001 was more stable at higher pH values, i.e., around pH 8.0. The objective of the current study was to further optimize VMX-C001 formulations using material stressed under a variety of conditions. A total of three different arginine / glutamic acid buffered VMX-C001 formulations were subjected to stress conditions (-80°C, 2-8°C, and 25°C / 60%RH), freeze / thaw cycling, and gentle shaking with the intention of selecting a formulation most suitable for the liquid bulk drug substance and filled drug product.

[0183] Turbidity was determined at A330 nm. Osmolality was determined on a Vapor Pressure Osmometer (Wesco VAPRO® vapor pressure osmometer Model 5600). Viscosity was determined at a m-VROC Viscometer (Rheosense). Iso-aspartic acid residue determination was performed by enzyme-linked reversed phase HPLC on a ISOQUANT AM- 1771 AGO platform.

[0184] A total of three arginine / glutamic acid buffered formulations of 10 mg / mL VMX-C001 were prepared at pH 8.0 (Table 3). All components were dissolved in water except for PS80, and then pH titrated using 5N NaOH. All the buffers were filtered through a 0.2 pm PES membrane before usage. A volume of 0.4 mL of test sample was aseptically placed in 2 mL glass vials that were subsequently stoppered. VMX-C001Ax08 was placed into pre-hydrated 10K MWCO dialysis cassettes and dialyzed at 2-8°C against IL of each buffer to be evaluated (3 changes of 1 liters). Total dialysis time of 1.5 days. The dialyzed material was subsequently concentrated by centrifugation to achieve a target concentration of >10 mg / mL. All test samples were then diluted with the appropriate buffers to a target concentration of 10 mg / mL VMX-C001. Polysorb-ate-80 was added to corresponding formulations to achieve a final concentration of 0.01% (w / v). Formulations were filled the same day as recovery and concentrating. Under aseptic conditions, the resulting VMX-C001 formulations were filtered through a 0.2 pm PVDF membraneand then the liquid samples were dispensed into pre-sterilized borosilicate vials (0.4 mL fill in 2 mL vial), stoppered and crimped with aluminum seals. Vials were then placed under stress conditions for stability testing.

[0185] Table 3. Initial VMX-C001 liquid twelve week stability study formulations. All formulation contained 0.01% PS80.

[0186] ID# Buffer Cone pH Glycine Sorbitol Mannitol Sucrose system (mg / mL) (mM) (%) (%) (%) 17 10 mM 10 8.0 5

[0187] 18 arginine, 10 8.0 9

[0188] 19 10 mM 10 8.0 50 1.6 4

[0189] glutamic

[0190]

[0191] acid

[0192] Following stress incubation, the samples visual appearance, protein concentration, turbidity, and pH were noted. Sample pH values post dialysis were significantly less than dialysis buffer concentrations likely due to Donnan effects observed during diafiltration of acidic proteins in which charged particles near a semi-permeable membrane fail to distribute evenly across the two sides of the membrane. Fluctuations in pH were also seen throughout the study, with increases from measured at t = 0 of up to 0.3 pH units. Protein concentration was consistent with t=0 values following the different stress conditions. Visual appearance remained clear, colorless and with no visible particulates for all the formulations throughout the course of the study. Turbidity values were similar among the three formulations following the different stress conditions, although Formulations 18 and 19 showed a slight increase in turbidity over time at 2-8 °C, when compared to Formulation 17.

[0193] Stability data is presented graphically in Figures 4 and 5. All three formulations showed a similar decrease in main peak purity < 2.0% following 12 weeks storage at -80°C. A shoulder of the main SEC peak was observed following one week storage at 25°C / 60%RH and at t=8 weeks for samples stored at 2-8°C, which was also detected at later time points (data not shown). After 12 weeks storage at 2-8°C, all formulations showed 1.8% - 2.1% degradation, with Formulation #19 (containing glycine / mannitol / sucrose) showing a slightly greater decrease in purity as compared to the other formulations. After 4 weeks storage at 25°C / 60%RH, Formulation #17 (containing 5% sorbitol) and Formulation #18 (containing 9%sucrose) showed comparable degradation (-8.5% decrease in main peak) and whereas Formulation #19 saw a -10% decrease in main peak area.

[0194] Example 5

[0195] The determination of iso-aspartic acid residues uses reversed phase HPLC (RP-HPLC) to quantify the concentration of isoaspartic acid residues in proteins and peptides, specifically the global formulation of isoaspartic acid residues at exposed Asn and Asp sites, using the ISOQUANT® isoaspartate detection kit (Promega). Isoaspartic acid residues can result from the rearrangement of aspartic acid residues or the deamidation of asparagine residues. The presence of isoaspartic acid residues in a target protein can be determined by using the enzyme protein isoaspartyl methyltransferase (PIMT). PIMT works by catalyzing the transfer of a methyl group from S-adenosyl_L-methionine (SAM) to isoaspartic acid which generates S-adenosyl homocysteine (SAH), a coproduct of the methylation reaction. SAH concentrations quantified by RP-HPLC correlate directly to isoaspartic acid residues. Sample preparation consists of combining 10 pL of purified water, Reaction 5x Buffer, SAM stock solution, sample, and PIMT. This solution is then mixed thoroughly and incubated at 30°C for 30 minutes. A volume of 10 pL of the Stop Solution NR (6X solution) 0.3M phosphoric acid) is then added, and the samples are placed on ice or at -20°C in the dark for 10 minutes to stop the reaction. For a standard linear calibration curve, six SAH dilutions are prepared using purified water and SAH solution. Chromatography conditions include a flow rate of 1.0 mg / mL, a run time of 13 minutes, a mobile phase gradient of 50 mM potassium phosphate at pH 6.8 and HPLC grade methanol, and a Synergi 4 pm Hydro-RP 80 A (4.6 x 150 mm) column to quantify the concentration of isoaspartic acid residues by detecting the PIMT methylation reaction coproduct, SAH.

[0196] Detection is by UV at 260 nm.

[0197] As is shown in Figure 4C, a small increase in isoaspartate levels was detected in Formulation #17 (containing 5% sorbitol) as compared to the other formulations upon 4-week storage at 25°C / 60%RH (22.6% to 25.4%, respectively). After 12 weeks storage at 2-8°C, Formulation #17 also showed the lowest increase in deamidation as compared to the other formulations (3.3% vs. 3.9%; Figure 4D), whereas after 12 weeks of storage at -80°C all formulations had isoaspartate content comparable to t=0 (data not shown).Formulations 17, 18 and 19 were examined by size exclusion chromatography (also known as gel filtration chromatography), which separates the molecular forms of a protein based primarily on their hydrodynamic radius. In this method, larger molecular species (e.g., aggregates, IgG dimers and oligomers) elute earlier than the desired (monomeric) IgG species as pre-peaks. Smaller molecular species (e.g., degradation products and fragments) elute later as post-peaks. SE-UPLC was performed on the VMX-C001 stability samples using a Agilent AdvanceBio SEC 200A 1.9pm, 4.6x300 mm column and 10 mM Phosphate, 300 mM Arginine-HCl, pH 6.8 mobile phase.

[0198] The data are shown in Figure 5. All three formulations showed a similar decrease in main peak purity < 2.0% following 12 weeks storage at -80°C (Figure 5A). A shoulder of the main SEC peak was observed following one week storage at 25°C / 60%RH and at t=8 weeks for samples stored at 2-8°C, which was also detected at later time points (data not shown). After 12 weeks storage at 2-8°C, all formulations showed 1.8% - 2.1% degradation, with Formulation #19 (containing glycine / mannitol / sucrose) showing a slightly greater decrease in purity as compared to the other formulations (Figure 5B). After 4 weeks storage at 25°C / 60%RH, Formulation #17 (containing 5% sorbitol) and Formulation #18 (containing 9% sucrose) showed comparable degradation (-8.5% decrease in main peak) and whereas Formulation #19 saw a -10% decrease in main peak area.

[0199] Example 6

[0200] The objective of the study described herein was to examine the pH range of a lead VMX-C001 formulation composed of 10 mM arginine, 10 mM glutamic acid, 5% sorbitol, 0.01% PS80, pH 8.0, at two different VMX-C001 concentrations (10 mg / mL and 5 mg / mL) while increasing the amount of surfactant to 0.03% PS80. Moreover, as the protein of interest is a coagulation factor, the effects of addition of calcium chloride was also evaluated.

[0201] A total of five different arginine / glutamic acid buffered VMX-C001 formulations were prepared and subsequently assessed for stability upon storage at -80°C over a 12-week timeframe, with the intention of selecting a formulation most suitable for the liquid bulk drug substance and filled drug product.

[0202] Table 4. Formulations examined in the VMX-C001 liquid twelve week stability.Formula Buffer Cone pH 5 mM Sorbitol 0.03% tion ID system (mg / mL) CaCl2(%) PS80

[0203] 20 10 mM 10 8.0 - 5 +

[0204] 21 arginine, 10 7.5 - 5 +

[0205] 22 10 mM 5 8.0 + 5 +

[0206] 23 glutamic 5 7.5 - 5 +

[0207]

[0208] 24 acid 5 7.5 - 5 +

[0209] Formulation buffers were prepared according to Table 4. Briefly, all components were dissolved in water except for PS80, and then pH titrated using 5N NaOH. All buffers were filtered through a 0.2 pm PES membrane before usage.

[0210] VMX-C001Ax08 was concentrated to 13.4 mg / mL before being split and placed into pre-hydrated 10K MWCO dialysis cassettes and dialyzed at 2-8°C against IL of each buffer to be evaluated (3 changes of 1 liters). Total dialysis time of 1.5 days. The dialyzed material was recovered above the target concentration of 10 mg / mL. All test samples were then diluted with the appropriate buffers to a target concentration of 5 mg / mL or 10 mg / mL VMX-C001. Polysorbate -80 was added to corresponding formulations to achieve a final concentration of 0.03% (w / v).

[0211] Formulations were filled the same day as recovery and concentrating. Under aseptic conditions, the resulting VMX-C001 formulations were filtered through a 0.2 pm PVDF membrane and then the liquid samples were dispensed into presterilized borosilicate vials (0.5 mL fill in 2 mL vial), stoppered and crimped with aluminum seals. Vials were then placed under stress conditions for stability testing. Additional formulated material was retained for the lyophilized twelve week formulation study -round 2 (See Section 10).

[0212] Each test sample appeared as clear, colorless solutions with osmolalities ranging from 306 -317 mOsm.

[0213] Following incubation, the samples visual appearance, protein concentration, turbidity, and pH were noted as in Table 5. Sample pH values post dialysis were significantly less than dialysis buffer concentrations, in particular for the formulation containing 5 mM calcium chloride. No difference was observable in regards to protein concentration following the different time points. Visual appearance remained clear, colorless and with no visible particulates for all the formulations following each time point. Turbidity values were similar among the five formulations following the different time points.Table 5. Concentration, osmolality, and pH values for VMX-C001 liquid twelve week stability study -Round 2 formulations at t=0..

[0214] Formula pH Measured Cone. Osmolality tion ID pH (mg / mL) (mOsm / kg) 20 8.0 7.5 10.4 310

[0215] 21 7.5 7.1 10.5 306

[0216] 22 8.0 7.4 5.2 310

[0217] 23 7.5 7.0 5.2 307

[0218]

[0219] 24 7.5 6.6 5.4 317

[0220] The resulting 5 mg / mL and 10 mg / mL VMX-C001 formulations were clear and colorless upon preparation. Each formulation behaved similarly following the different time points as all the appearance and concentration results were comparable to t=0 and the pH and turbidity value changes were similar among the formulations.

[0221] Following 12 weeks of storage at -80°C for all of the various formulation configurations, the amount of HMW was consistent with t=0 values, whereas overall sample purity decreased by roughly 1% due to an increase of a LMW in all formulations. See Table 6. The lack of change in the amount HMW result differs from the previous 12-week study in which the formulations contained only 0.01% PS80 that resulted in a 1% increase in HMW after 12 weeks storage at -80°C (See Section 7.4). Moreover, the results of the current study indicate there are no differences in VMX-C001 stability as a function of concentration (10 mg / mL vs. 5 mg / mL), pH value (pH 8.0 vs. pH 7.5), nor in the presence of 5 mM calcium chloride.

[0222] Table 6. Tabular results of SEC for VMX-C001 liquid twelve week stability study formulations.

[0223] SE-UPEC (% purity)

[0224] HMW MAIN PEAK*

[0225] Formulation # LMW

[0226] -80 °C

[0227] T=0

[0228] 4 weeks 8 weeks 12 weeks 20 0.5 0.6 0.8 0.5

[0229] 96.9 97.2 96.3 95.9 2.5 2.2 2.9 3.6

[0230]

[0231] 21 0.5 0.6 0.8 0.597.0 97.1 96.2 95.9

[0232] 2.4 2.3 3.0 3.6 22 0.6 0.6 0.8 0.5

[0233] 97.0 97.2 96.2 96.0 2.4 2.2 3.0 3.5 23 0.6 0.6 0.8 0.5

[0234] 96.9 97.2 96.1 96.0 2.5 2.2 3.1 3.5 24 0.7 0.8 1.0 0.6 96.7 97.0 96.1 96.0

[0235]

[0236] 2.5 2.2 2.9 3.4 * Main peak includes relative area for main peak and main peak shoulder

[0237] Following 12 weeks of storage at -80°C for all of the various formulation configurations, the amount of HMW was consistent with t=0 values, whereas overall sample purity decreased by roughly 1% due to an increase of a LMW in all formulations. The lack of change in the amount HMW result differs from the previous 12-week study in which the formulations contained only 0.01% PS80 that resulted in a 1% increase in HMW after 12 weeks storage at -80 °C. Moreover, the results of the current study indicate there are no differences in VMX-C001 stability as a function of concentration (10 mg / mL vs. 5 mg / mL), pH value (pH 8.0 vs. pH 7.5), nor in the presence of 5 mM calcium chloride.

[0238] After 12 weeks of storage at -80°C, a less than 1% increase in deamidation levels was observed regardless of the formulation configuration (data not shown). This result differs from the earlier stability study in which a corresponding 10 mg / mL VMX-C001 pH 8.0 formulation containing only 0.01% PS80 displayed deamidation levels consistent with the t=0 value.

[0239] Example 7

[0240] A total of five arginine / glutamic acid buffered formulations of 10 mg / mL VMX-C001 were prepared as described in Table 7. A volume of 0.5 mL of test sample was aseptically placed in 2 mL glass vials that were subsequently lyophilized, stoppered, and sealed.

[0241] Briefly, all the components were dissolved in water except for PS80, and then pH titrated using 5N NaOH. All buffers were filtered through a 0.2 pm PES membrane before usage.VMX-C001Ax08 was concentrated to 13.4 mg / mL before being split and placed into pre-hydrated 10K MWCO dialysis cassettes and dialyzed at 2-8°C against IL of each buffer to be evaluated (3 changes of 1 liters). Total dialysis time of 1.5 days. The dialyzed material was recovered above the target concentration of 10 mg / mL. All test samples were then diluted with the appropriate buffers to a target concentration of 10 mg / mL VMX-C001. Polysorb ate -80 was added to corresponding formulations to achieve a final concentration of 0.01% (w / v). Formulations were filled the same day as recovery and concentrating. Under aseptic conditions, the resulting VMX-C001 formulations were filtered through a 0.2 pm PVDF membrane and then the liquid samples were dispensed into pre-sterilized borosilicate vials (0.5 mL fill in 2 mL vial), lyophilized, stoppered, and crimped with aluminum seals. The resulting lyophilized cakes were white, glossy, and intact following a 58 hour freeze drying cycle. Vials were then placed under stress conditions for stability testing.

[0242] Table 7. Concentration, osmolality, and pH values for VMX-C001 liquid twelve week stability study -Round 2 formulations at t=0..

[0243] Formula Buffer system pH 5 mM Sorbitol Sucrose 0.01% tion ID CaCl2(%) (%) PS80 25 8.0 5 + 26 10 mM arginine, 7.5 5 + 27 10 mM glutamic 7.5 + 5 + 28 acid 8.0 9 +

[0244]

[0245] 29 7.5 9 +

[0246] Reconstitution of the lyophilized cake products with 0.5 mL of sterile water for injection (sWFI) resulted in approximately a 10 mg / mL VMX-C001 concentration and osmolality values ranging from 310 to 326 mOsm / kg (data not shown).

[0247] Structurally, both the sorbitol and the sucrose formulations remained intact after four weeks at 2-8°C, however, however after t=4 weeks storage at 25°C / 60% RH melting of all of the sorbitol containing formulations was observed (data not shown). At the t=8 weeks timepoint the cake products of the sorbitol formulations stored at 2-8°C began to collapse. Reconstitution times of the collapsed cakes increased, reaching up to two minutes prior to complete dissolution for the sorbitol containing formulations. Of note, the moisture content of the cake products ofsucrose containing formulations increased from roughly 2% to roughly 8% after 12 weeks storage at 25°C / 60% RH, whereas the residual moisture of the sorbitol containing formulations was double from roughly 1.5% to 3% at the t=12 week timepoint.

[0248] The pH (albeit the measured sample pH is lower than the dialysis buffer due to Donnan effects), concentration, turbidity and visual appearance (clear, colorless, no particles) of the reconstituted VMX-C001 material was consistent throughout the study (Table 8).

[0249] Reconstitution of the lyophilized cake products with 0.5 mL of sterile water for injection (sWFI) resulted in approximately a 10 mg / mL VMX-C001 concentration and osmolality values ranging from 310 to 326 mOsm / kg. The pH, concentration, turbidity and visual appearance (clear, colorless, no particles) of the reconstituted VMX-C001 material was within assay variability throughout the study for the sucrose containing VMX-C001 formulations, whereas a bit more variability was observed, particularly in the turbidity measurement, for the sorbitol containing VMX-C001 formulations likely due to the varying degree of cake collapse.

[0250] Structurally, both the sorbitol and the sucrose formulations remained intact after four weeks at 2-8°C, however, however after t=4 weeks storage at 25°C / 60% RH melting of all of the sorbitol containing formulations was observed. At the t=8 weeks timepoint the cake products of the sorbitol formulations stored at 2-8°C began to collapse. Reconstitution times of the collapsed cakes increased, reaching up to two minutes prior to complete dissolution for the sorbitol containing formulations stored at 25°C / 60% RH. These results strongly suggest the need for additional lyophilization cycle development for the sorbitol containing formulations. Of note, the moisture content of the cake products of sucrose containing formulations increased from roughly 2% to roughly 8% after 12 weeks storage at 25°C / 60% RH, whereas the residual moisture of the sorbitol containing formulations was double from roughly 1.5% to 3% at the t=12 week timepoint. Note, observable moisture increases inside the lyophilized vials can be caused by moisture transfer from the stoppers, or over the long-term, moisture increase maybe caused by external moisture permeation through the stoppers. The increase in moisture is also dependent upon the hygroscopic nature of the sugars present in the formulation.Iso-aspartate levels were consistent with t=0 after 12 weeks storage at 2-8°C. However, after 12 weeks at 25°C / 60% RH increases in the levels of isoaspartate were observed for both the sorbitol and the sucrose containing formulations as compared to T=0 (1.4 % to 3.5% ) The sucrose containing formulations prepared at pH 8.0 (actual sample pH 7.8) did not show changes in deamidation levels after 12-weeks storage at 25°C / 60% RH, whereas samples prepared at pH 7.5 (actual sample pH 7.3) increases are being observed.

[0251] Overall, all formulations are showing equivalent stabilities. The addition of CaCk did not impart additional stability as compared to the similar formulation not containing CaC12. Increases in deamidation levels suggest that the lyophilized formulations should be stored at 2-8°C as opposed to 25°C

[0252] Example 8

[0253] Formulation #17 (10 mM arginine, 10 mM glutamic acid, 5% sorbitol, 0.01% PS80, pH 8.0) was selected as the lead formulation to facilitate the manufacturing engineering run as well as complete method qualification.

[0254] Overall evaluation of the stability data shown in Tables 10 and 11 indicates that this formulation is within specification for at least up to 28 months at the long term storage condition at -20°C ± 5°C (Table 10) and up to 6 months at the accelerated storage condition at 25°C ± 3°C (Tables 10 and 11). The overall stability data indicates that the deviations raised in the stability study did not have any overall impact.Table 8. Tabular results for VMX-C001 lyophilized twelve week study formulations.

[0255] A280 concentration (mg / mL)

[0256] Turbidity (A330)

[0257] Cake visual appearance*

[0258] Reconstitution time (sec)

[0259] Formulation

[0260] Liquid visual appearance**

[0261] # pH

[0262] Moisture content (%)

[0263] 2-8°C 25°C / 60% RH

[0264] T=0

[0265] 4 weeks 8 weeks 12 weeks 4 weeks 8 weeks 12 weeks

[0266] 25 10.3 10.4 10.3 9.9 10.7 10.1 10.0

[0267] 0.050 0.077 0.052 0.044 0.062 0.069 0.077

[0268] I, G I, G G, M G, M M M M

[0269] 17 15 37 27 96 108 130

[0270]

[0271] C, NC, NP C, NC, NP C, NC, NP C, NC, NP C, NC, NP C, NC, NP C, NC, NP

[0272] 7.4 7.3 7.5 7.3 7.3 7.3 7.3

[0273] 1.4 NT NT 2.9 NT NT 3.4

[0274] 26 10.1 10.1 10.3 9.8 10.2 10.1 10.1

[0275] 0.048 0.051 0.050 0.060 0.062 0.063 0.073

[0276] I, G I, G G, M G, M M M M

[0277] 17 19 40 33 102 112 113

[0278] C, NC, NP C, NC, NP C, NC, NP C, NC, NP C, NC, NP C, NC, NP C, NC, NP

[0279] 6.7 6.8 7.0 6.8 6.8 6.8 6.8

[0280] 1.4 NT NT 1.5 NT NT 2.7

[0281] 27 10.1 10.1 10.4 10.0 10.1 10.1 10.0

[0282] 0.066 0.068 0.069 0.069 0.073 0.080 0.093

[0283] I, G I, G G, M G, M M M M

[0284]

[0285] 31 24 31 31 99 99 137C, NC, NP C, NC, NP C, NC, NP C, NC, NP C, NC, NP C, NC, NP C, NC, NP

[0286] 6.6 6.5 6.4 6.4 6.6 6.4 6.5

[0287] NT NT NT NT NT NT 2.8

[0288] 28 9.8 9.9 9.8

[0289] 9.8 9.9 10.0 9.8 0.054 0.057 0.054

[0290] 0.070 0.053 0.058 0.054 I, G I, G I, G

[0291] I, G I, G I, G I, G 31 38 48

[0292] 38 22 28 36 C, NC, NP C, NC, NP C, NC, NP NC, NP C, NC, NP C, NC, NP C, NC, NP 7.3 7.3 7.3

[0293] 7.4 7.3 7.3 7.3 NT NT 7.6

[0294] 2.6 * NT NT 2.4

[0295] 29 9.8 9.8 9.8 10.0 9.7 9.9 9.6

[0296] 0.056 0.056 0.063 0.060 0.061 0.063 0.069

[0297] I, G I, G I, G I, G I, G I, G I, G

[0298] 29 29 19 24 31 36 51 C, NC, NP C, NC, NP C, NC, NP C, NC, NP C, NC, NP C, NC, NP C, NC, NP

[0299]

[0300] 7.3 7.3 7.3 7.3 7.3 7.3 7.3

[0301]

[0302] 2.1 2.1 NT NT 2.5 NT 7.9 STT=Not Tested.

[0303] * I=Intact; D=Detached; M=melt back; G=Glossy

[0304] ** C=Clear; NC=No Color; NP=No Particles; P=Very Few Particles

[0305] ¥ Form Buffer 28 Placebo used for T=0 moisture assay

[0306] E= starting pH of dialysis buffer was actually 7.5Table 9. Tabular results of SEC for VMX-C001 lyophilized twelve week study formulations.

[0307] SE-UPLC (% Purity)

[0308] HMW

[0309] Formulation Main Peak

[0310] LMW

[0311] #

[0312] 2-8°C 25°C / 60% RH

[0313] T=0

[0314] 4 weeks 8 weeks 12 weeks 4 weeks 8 weeks 12 weeks

[0315] 25 0.7 0.5 0.7 0.5 0.9 1.0 1.0

[0316] 95.4 96.3 96.2 95.7 96.1 96.1 95.5

[0317] 3.9 3.2 3.1 3.7 3.0 2.9 3.5

[0318] 26 0.7 0.5 0.8 0.6 0.8 1.0 1.0

[0319] 95.4 96.3 96.1 95.8 96.3 96.0 95.4

[0320] 3.9 3.2 3.1 3.7 2.9 3.0 3.7

[0321] 27 0.9 0.7 0.8 0.7 1.0 1.1 1.2

[0322]

[0323] 95.3 96.1 96.5 95.7 96.1 95.9 95.4

[0324] 3.8 3.2 2.8 3.6 2.9 3.0 3.4

[0325] 28 0.6 0.6 0.7 0.5 0.6 0.7 0.6

[0326] 95.5 96.3 96.4 95.9 96.6 96.4 95.9

[0327] 3.9 3.1 2.9 3.5 2.8 2.9 3.5

[0328] 29 0.7 0.5 1.0 0.5 0.6 0.6 0.6

[0329] 95.8 96.9 96.0 95.9 96.5 96.5 96.0

[0330]

[0331] 3.6 2.7 3.0 3.6 2.9 2.8 3.4Table 10. Stability study for formulation #17.

[0332] Observation (months)

[0333] Feature Storage

[0334] Time 0 1 3 6 12 18 28

[0335] -20 °C < STD1 < STD1 < STD1 < STD1 < STD1 < STD1

[0336] Clarity < STD1

[0337] +5 °C < STD1

[0338] -20 °C Essentially Essentially Essentially Essentially Essentially Essentially Appearance

[0339] Essentially free free free free free free

[0340] visible

[0341] +5 °C free Essentially Essentially

[0342] particles

[0343] free free

[0344] -20 °C 7.4 7.4 7.4 7.5 7.5 7.6

[0345] pH 7.6

[0346] +5 °C 7.4 7.5

[0347] Cone -20 °C 10.4 10.4 10.4 10.4 10.4 10.4

[0348]

[0349] 10.3

[0350] (mg / mL) +5 °C 10.4 10.4

[0351] -20 °C 96.4 96.4 97.1 97.0 95.3 96.1

[0352] SE-UHPLC

[0353] 96

[0354] main peak +5 °C 96.4 96.3 96.5

[0355] Clotting -20 °C 8 8 9 8 7 8

[0356] 8

[0357] lU / mg +5 °C 7 7 8

[0358]

[0359] Table 11. Stability study for formulation #17.

[0360] Observation time

[0361] Feature per

[0362] container Storage 0 2 weeks 1 M 3 M 6 M 9 M 12 M

[0363] Sub -visible +5 °C 6 2

[0364] particles >25 18

[0365] +25 °C

[0366] pm <1 1

[0367] Sub -visible +5 °C 41 13

[0368] particles >10 +25 °C 168

[0369] pm 25 16

[0370] Sub -visible +5 °C 61 19

[0371] particles > 9 +25 °C 215

[0372] pm 37 23

[0373]

[0374] Sub -visible +5 °C 203 93 particles > 5 656

[0375] +25 °C

[0376] pm 249 134

[0377] Osmolality +5 °C 306 309

[0378] 306

[0379] mOsmol / kg +25 °C 307

[0380] +5 °C 7.7 7.7 7.6 7.7 7.6

[0381] pH 7.6

[0382]

[0383] +25 °C 7.7 7.7 7.6 7.6SEQ ID NO:1 Sequence of human coagulation Factor X (UniProt P00742) 1 mgrplhlvll saslagllll geslfirreq annilarvtr ansfleemkk ghlerecmee 61 tcsyeearev fedsdktnef wnkykdgdqc etspcqnqgk ckdglgeytc tclegfegkn 121 celftrklcs Idngdcdqfc heeqnsvvcs cargytladn gkaciptgpy pcgkqtlerr 181 krsvaqatss sgeapdsitw kpydaadldp tenpfdlldf nqtqpergdn nltrivggqe 241 ckdgecpwqa llineenegf cggtilsefy iltaahclyq akrfkvrvgd rnteqeegge 301 avhevevvik hnrftketyd fdiavlrlkt pitfrmnvap aclperdwae stlmtqktgi 361 vsgfgrthek grqstrlkml evpyvdrnsc klsssfiitq nmfcagydtk qedacqgdsg 421 gphvtrfkdt yfvtgivswg egcarkgkyg iytkvtaflk widrsmktrg Ipkakshape 481 vitssplk

[0384] SEQ ID NO: 2 (Light chain of VMX-C001)

[0385] 1 ansfleemkk ghlerecmee tcsyeearev fedsdktnef wnkykdgdqc etspcqnqgk 61 ckdglgeytc tclegfegkn celftrklcs Idngdcdqfc heeqnsvvcs cargytladn 121 gkaciptgpy pcgkqtler 139

[0386] SEQ ID NO 3 (Heavy chain of VMX-C001)

[0387] 1 ivggqeckdg ecpwqallin eenegfcggt ilsefyilta ahclyqakrf kvrvgdrnte 61 qeeggeavhe vevvikhKKF VPPQKAYKFD LAAydfdiav Irlktpitfr mnvapaclpe 121 rdwaestlmt qktgivsgfg rthekgrqst rlkmlevpyv drnscklsss fiitqnmfca 181 gydtkqedac qgdsggphvt rfkdtyfvtg ivswgegcar kgkygiytkv taflkwidrs 241 mktrglpkak shapevitss plk

Claims

Claims1. A formulation of a F actor X, comprising5-20 mg / mL of a Factor X,0.05-5 mg / mL of a non-ionic surfactant,5-150 mg / mL of a stabilizer, and0.5-10 mg / mL of a buffering agent, the formulation having a pH of 7-9, wherein said Factor X is a zymogen Factor X.

2. The formulation of claim 1, comprising about 10 mg / mL of a Factor X.

3. The formulation of claim 1 or claim 2, wherein the Factor X, after activation, has a specific activity of 1-100 IU / mg, such as 1-50 IU / mg.

4. The formulation of any one of claims 1-3, wherein the stabilizer is a non-metal salt, a sugar, a sugar alcohol, and / or an amino acid.

5. The formulation of any one of claims 1-4, wherein the stabilizer is sorbitol.

6. The formulation of any one of claims 1-5, wherein the non-ionic surfactant is or comprises a polysorbate.

7. The formulation of any one of claims 1-6, wherein the non-ionic surfactant is or comprises polyoxyethylene (20) sorbitan monooleate.

8. The formulation of any one of claims 1-7, comprising 0.1 mg / mL of a non-ionic surfactant.

9. The formulation of any one of claims 1-8, comprising 50 mg / mL of a stabilizer.

10. The formulation of any one of claims 1-9, wherein the buffering agent is or comprises L- arginine and L-glutamic acid.

11. The formulation of any one of claims 1-10, wherein the buffering agent is 1.72 mg / mL L- arginine and 1.45 mg / mL L-glutamic acid.

12. The formulation of any one of claims 1-11, comprising a recombinant Factor X.

13. The formulation of any one of claims 1-12, wherein the Factor X is obtained from a eukaryotic cell-based expression system, preferably VMX-C001.

14. A lyophilized composition that provides the formulation of any one of claims 1-13 after reconstitution.

15. The formulation of any one of claims 1-13, for use as a medicament.

16. The formulation for use of claim 15, whereby the medicament comprises 150 mg of Factor X.

17. The formulation for use of claim 15 or 16, whereby the medicament comprises 15 mL of the formulation.

18. The formulation for use according to any one of claims 15-17, whereby the medicament is administered by injection.

19. The formulation for use of claim 18, whereby the medicament is administered by intravenous injection.

20. The formulation for use of claim 18 or claim 19, whereby the medicament is administered by intravenous slow push injection.

21. The formulation for use according to any one of claims 18-20, whereby the medicament is administered by intravenous slow push injection over 10 seconds.

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