Preparation of factor XA derivatives

Abstract

Preparation of Factor XA Derivatives.SOLUTION: The present disclosure provides methods for large-scale manufacturing of fXa derivative proteins to obtain high yields of high purity protein products. The method can comprise adding a detergent to a sample comprising a polynucleotide construct encoding a protein and purifying the protein via soybean trypsin inhibitor (STI) - based affinity chromatography, ion exchange and mixed mode chromatography, and hydrophobic interaction.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference to related patent applications This application claims the benefit of U.S. Provisional Patent Application Serial No. 62 / 351,841, filed June 17, 2016, the contents of each of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Modified derivatives of factor Xa (fXa) protein have been developed that are useful as antidotes to anticoagulants that target fXa. These derivatives are being developed as general reversal agents for patients undergoing anticoagulation therapy with oral or injectable factor Xa inhibitors who require reversal of anticoagulation. Summary of the Invention

[0003] The present disclosure provides methods for large-scale production of fXa antidote polypeptides to obtain high yields of highly purified protein product. In one embodiment, a method is provided for preparing a polypeptide product expressed from a polynucleotide construct comprising a nucleic acid sequence encoding the nucleic acid sequence of SEQ ID NO:7 or an amino acid sequence having at least 90% sequence identity to the amino acid sequence encoded by SEQ ID NO:7. The method may include adding a detergent to a sample containing the polynucleotide construct and purifying the encoded antidote protein via soybean trypsin inhibitor (STI)-based affinity chromatography, ion exchange and mixed-mode chromatography, and / or hydrophobic interaction.

[0004] In one embodiment, a method is provided for preparing a polypeptide product expressed from a polynucleotide construct comprising the nucleic acid sequence of SEQ ID NO:7 or a nucleic acid sequence encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequence encoded by SEQ ID NO:7, comprising adding a detergent to a sample comprising the polynucleotide construct and the polypeptide product expressed from the polynucleotide construct; loading the sample onto a soybean trypsin inhibitor (STI)-based affinity chromatograph and eluting the polypeptide with a first elution buffer to produce a first eluted sample, wherein the loaded sample does not contain organic solvent; loading the first eluted sample onto an ion exchange and mixed-mode chromatograph and eluting the polypeptide with a second elution buffer comprising at least 1 M inorganic salt to produce a second eluted sample; and loading the second eluted sample onto a hydrophobic interaction chromatograph and eluting the polypeptide with a third elution buffer comprising at least 2 mM sodium chloride, thereby preparing a purified sample comprising the polypeptide product.

[0005] In some embodiments, the surfactant comprises Triton X-100 (polyethylene glycol P-(1,1,3,3-tetramethylbutyl)-phenyl ether). In some embodiments, the first elution buffer comprises 0.5 M to 2 M arginine. In some embodiments, the pH of the first elution buffer is about 5 to 5.4. In some embodiments, the ion exchange and mixed-mode chromatograph comprises a ceramic hydroxyapatite Type I chromatograph.

[0006] In some embodiments, the second elution buffer comprises at least 2 M inorganic salt. In some embodiments, the inorganic salt is sodium chloride. In some embodiments, the hydrophobic interaction chromatograph comprises an octyl sepharose chromatograph.

[0007] In some embodiments, the method further comprises a purification step on an anion exchange chromatograph, hi some embodiments, the anion exchange chromatograph comprises a Sartobind™ ion exchange membrane.

[0008] The method further comprises subjecting one or more of the samples to filtration through a nanofleece filter. In some embodiments, filtration through a nanofleece filter precedes loading the sample onto an STI-based affinity chromatograph.

[0009] In some embodiments, the purified sample contains less than about 1% of contaminating proteins not expressed from the polynucleotide construct. In some embodiments, the polypeptide product is expressed in cells comprising the polynucleotide construct. In some embodiments, the cells are grown in culture under conditions to produce at least 100 mg of polypeptide product per liter of culture. In some embodiments, the cells are grown in culture under conditions to produce at least 200 mg of polypeptide product per liter of culture. In some embodiments, the purified sample comprises more than about 50% of the polypeptide product produced in the culture. In some embodiments, the purified sample comprises more than about 100 mg of polypeptide product from the production amount per liter of culture.

[0010] In some embodiments, the polypeptide product is a double-chain polypeptide comprising a light chain and a heavy chain. In some embodiments, about 20% to 50% of the polypeptide products in a purified sample have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 5. In some embodiments, about 5% to 95% of the heavy chains consisting of the amino acid sequence of SEQ ID NO: 5 have two O-linked glycosylations, and about 5% to 95% of the heavy chains consisting of the amino acid sequence of SEQ ID NO: 5 have one O-linked glycosylation. In some embodiments, about 40% to 80% of the polypeptide products in a purified sample have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8. In some embodiments, at least about 90% of the heavy chains consisting of the amino acid sequence of SEQ ID NO: 8 have one O-linked glycosylation. In some embodiments, about 2% to 12% of the polypeptide products in a purified sample have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 9. In some embodiments, about 0.1% to 1.5% of the polypeptide products in a purified sample have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 10. In some embodiments, about 2% to 8% of the polypeptide products in a purified sample have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 11. In some embodiments, about 35% to 60% of the polypeptide products in a purified sample have a light chain consisting of the amino acid sequence of SEQ ID NO: 4.

[0011] In one embodiment, a polypeptide prepared by the method of any one of the embodiments is also provided.

[0012] In one embodiment, a pharmaceutical composition is provided comprising a pharmaceutically acceptable carrier and a polypeptide portion of a double-chain polypeptide, wherein approximately 35% to 60% of the double-chain polypeptides have a light chain consisting of the amino acid sequence of SEQ ID NO: 4, approximately 20% to 60% of the double-chain polypeptides have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 5, approximately 40% to 60% of the double-chain polypeptides have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8, and less than 10% of the double-chain polypeptides have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 9.

[0013] In some embodiments, fewer than 5% of the double-chain polypeptides have heavy chains consisting of the amino acid sequence of SEQ ID NO: 9. In some embodiments, fewer than 3% of the double-chain polypeptides have heavy chains consisting of the amino acid sequence of SEQ ID NO: 9. In some embodiments, about 0.1% to 1.5% of the double-chain polypeptides have heavy chains consisting of the amino acid sequence of SEQ ID NO: 10. In some embodiments, about 2% to 8% of the double-chain polypeptides have heavy chains consisting of the amino acid sequence of SEQ ID NO: 11. In some embodiments, about 30% to 70% of the heavy chains consisting of the amino acid sequence of SEQ ID NO: 5 have two O-linked glycosylation. In some embodiments, about 30% to 70% of the heavy chains consisting of the amino acid sequence of SEQ ID NO: 5 have one O-linked glycosylation. In some embodiments, at least about 90% of the heavy chains consisting of the amino acid sequence of SEQ ID NO: 8 have one O-linked glycosylation.

[0014] In some embodiments, the formulation is lyophilized. In some embodiments, the composition further comprises L-arginine HCl or L-arginine acetate. In some embodiments, the composition further comprises sucrose. In some embodiments, the composition further comprises mannitol. Also provided in one embodiment is a method for reversing or inhibiting anticoagulation in a patient undergoing anticoagulation therapy with a factor Xa inhibitor, comprising administering to the patient a pharmaceutical composition according to any one of the embodiments of the present disclosure. In certain embodiments, for example, the following are provided: (Item 1) 1. A method for preparing a polypeptide product expressed from a polynucleotide construct comprising a nucleic acid sequence encoding the nucleic acid sequence of SEQ ID NO:7 or an amino acid sequence having at least 90% sequence identity to the amino acid sequence encoded by SEQ ID NO:7, comprising: adding a detergent to a sample comprising the polynucleotide construct and a polypeptide product expressed from the polynucleotide construct; loading the sample onto a soybean trypsin inhibitor (STI)-based affinity chromatograph and eluting the polypeptide with a first elution buffer to generate a first eluted sample, wherein the loaded sample is free of organic solvent; loading the first eluted sample onto an ion exchange and mixed-mode chromatograph and eluting the polypeptide with a second elution buffer comprising at least 1 M inorganic salt to produce a second eluted sample; loading the second eluted sample onto a hydrophobic interaction chromatograph and eluting the polypeptide with a third elution buffer comprising at least 2 mM sodium chloride; This method thereby prepares a purified sample containing the polypeptide product. (Item 2) 2. The method of claim 1, wherein the surfactant comprises Triton X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether). (Item 3) 3. The method according to item 1 or 2, wherein the first elution buffer contains 0.5 M to 2 M arginine. (Item 4) Item 4. The method according to Item 3, wherein the pH of the first elution buffer is about 5 to 5.4. (Item 5) Item 10. The method of any of the preceding items, wherein the ion exchange and mixed-mode chromatograph comprises a ceramic hydroxyapatite Type I chromatograph. (Item 6) 10. The method of any of the preceding items, wherein the second elution buffer comprises at least 2 M inorganic salt. (Item 7) 7. The method according to item 6, wherein the inorganic salt is sodium chloride. (Item 8) Item 10. The method of any of the preceding items, wherein the hydrophobic interaction chromatograph comprises an octyl sepharose chromatograph. (Item 9) Item 1. The method according to item 1, further comprising a purification step by anion exchange chromatography. (Item 10) 10. The method of claim 9, wherein the anion exchange chromatograph comprises a Sartobind™ ion exchange membrane. (Item 11) 10. The method of any of the preceding items, further comprising subjecting one or more of the samples to filtration through a nanofleece filter. (Item 12) 12. The method of claim 11, wherein the filtration through the nanofleece filter precedes loading the sample onto the STI-based affinity chromatograph. (Item 13) 10. The method of any of the preceding items, wherein the purified sample contains less than about 1% contaminating proteins not expressed from the polynucleotide construct. (Item 14) The method of any of the preceding items, wherein the polypeptide product is expressed in a cell containing the polynucleotide construct. (Item 15) 15. The method of claim 14, wherein the cells are grown in culture under conditions to produce at least 100 mg of the polypeptide product per liter of culture. (Item 16) 16. The method of claim 15, wherein the cells are grown in culture under conditions to produce at least 200 mg of the polypeptide product per liter of culture. (Item 17) 17. The method of claim 15 or 16, wherein the purified sample comprises more than about 50% of the polypeptide product produced in the culture medium. (Item 18) 17. The method of claim 15 or 16, wherein the purified sample comprises more than about 100 mg of the polypeptide product from a production amount per liter of the culture medium. (Item 19) Item 11. The method of any of the preceding items, wherein the polypeptide product is a double-chain polypeptide comprising a light chain and a heavy chain. (Item 20) 20. The method according to Item 19, wherein approximately 20% to 50% of the polypeptide product in the purified sample has a heavy chain consisting of the amino acid sequence of SEQ ID NO:5. (Item 21) 21. The method of claim 20, wherein about 5% to 95% of the heavy chain consisting of the amino acid sequence of SEQ ID NO: 5 has two O-linked glycosylation, and about 5% to 95% of the heavy chain consisting of the amino acid sequence of SEQ ID NO: 5 has one O-linked glycosylation. (Item 22) 22. The method according to any one of Items 19 to 21, wherein approximately 40% to 80% of the polypeptide products in the purified sample have a heavy chain consisting of the amino acid sequence of SEQ ID NO:8. (Item 23) 23. The method of claim 22, wherein the heavy chain consisting of at least about 90% of the amino acid sequence of SEQ ID NO: 8 has one O-linked glycosylation. (Item 24) 24. The method according to any one of Items 19 to 23, wherein approximately 2% to 12% of the polypeptide products in the purified sample have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 9. (Item 25) 25. The method according to any one of Items 19 to 24, wherein approximately 0.1% to 1.5% of the polypeptide product in the purified sample has a heavy chain consisting of the amino acid sequence of SEQ ID NO: 10. (Item 26) 26. The method according to any one of Items 19 to 25, wherein approximately 2% to 8% of the polypeptide product in the purified sample has a heavy chain consisting of the amino acid sequence of SEQ ID NO: 11. (Item 27) 27. The method according to any one of Items 19 to 26, wherein approximately 35% to 60% of the polypeptide products in the purified sample have a light chain consisting of the amino acid sequence of SEQ ID NO: 4. (Item 28) A polypeptide prepared by the method according to any one of items 1 to 27. (Item 29) 1. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polypeptide portion of a double-chain polypeptide, approximately 35% to 60% of the double-chain polypeptides have a light chain consisting of the amino acid sequence of SEQ ID NO: 4; approximately 20% to 60% of the double-chain polypeptides have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 5; approximately 40% to 60% of the double-chain polypeptides have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8; The pharmaceutical composition, wherein less than 10% of the double-chain polypeptides have a heavy chain consisting of the amino acid sequence of SEQ ID NO:9. (Item 30) 30. The pharmaceutical composition of item 29, wherein less than 5% of the double-chain polypeptides have a heavy chain consisting of the amino acid sequence of SEQ ID NO:9. (Item 31) 30. The pharmaceutical composition of claim 29, wherein less than 3% of the double-chain polypeptides have a heavy chain consisting of the amino acid sequence of SEQ ID NO:9. (Item 32) 32. The pharmaceutical composition according to any one of Items 29 to 31, wherein approximately 0.1% to 1.5% of the double-chain polypeptide has a heavy chain consisting of the amino acid sequence of SEQ ID NO:10. (Item 33) 33. The pharmaceutical composition according to any one of Items 29 to 32, wherein approximately 2% to 8% of the double-chain polypeptides have a heavy chain consisting of the amino acid sequence of SEQ ID NO:11. (Item 34) 34. The pharmaceutical composition according to any one of items 29 to 33, wherein the heavy chain consisting of about 30% to 70% of the amino acid sequence of SEQ ID NO: 5 has two O-linked glycosylations. (Item 35) 35. The pharmaceutical composition of any one of items 29 to 34, wherein the heavy chain consisting of about 30% to 70% of the amino acid sequence of SEQ ID NO: 5 has one O-linked glycosylation. (Item 36) 36. The pharmaceutical composition of any one of items 29 to 35, wherein the heavy chain consisting of at least about 90% of the amino acid sequence of SEQ ID NO: 8 has one O-linked glycosylation. (Item 37) 37. The pharmaceutical composition according to any one of items 29 to 36, wherein the formulation is lyophilized. (Item 38) 38. The pharmaceutical composition according to any one of items 29 to 37, further comprising L-arginine HCl or L-arginine acetate. (Item 39) 39. The pharmaceutical composition according to any one of items 29 to 38, further comprising sucrose. (Item 40) 40. The pharmaceutical composition according to any one of items 29 to 39, further comprising mannitol. (Item 41) 41. A method for reversing or inhibiting anticoagulation in a patient undergoing anticoagulation therapy with a factor Xa inhibitor, the method comprising administering to the patient the pharmaceutical composition according to any one of items 29 to 40. [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 illustrates a purification process 100 according to one embodiment of the present invention. [Figure 2] FIG. 2 illustrates a post-scale up process according to one embodiment of the present invention. [Figure 3] FIG. 3 illustrates the evaluation of three potential subsequent processes (DSPs) according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0016] definition The following description describes exemplary embodiments of the present technology, but it should be recognized that such description does not limit the scope of the present disclosure, but is provided as a description of exemplary embodiments.

[0017] All numerical designations, such as, for example, pH, temperature, time, concentration, and molecular weight, including ranges, are approximations that are subject to variation (+) or (-) by increments of 0.1 or 10%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the word "about." It is also to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents thereof are known in the art.

[0018] As used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a pharmaceutically acceptable carrier" includes a plurality of pharmaceutically acceptable carriers, including mixtures thereof.

[0019] As used herein, the term "comprising" is intended to mean that the compositions and methods include the recited elements, but do not exclude other elements. When used to define compositions and methods, "consisting essentially of" means that the combination does not include other elements that have an essential meaning for the intended use. Thus, a composition consisting essentially of the elements defined herein does not exclude trace amounts of contaminants from isolation and purification methods and pharmaceutically acceptable carriers, such as phosphate-buffered saline, preservatives, etc. "Consisting of" means not including more than trace amounts of other ingredients and substantial method steps for administering the compositions of the present disclosure. Embodiments defined by each of these transitional terms are within the scope of the present disclosure.

[0020] The terms "protein" and "polypeptide" are used interchangeably in their broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In alternative embodiments, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may comprise a protein's or peptide's sequence. As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acid, including glycine and both the D and L optical isomers, amino acid analogs, and peptidomimetics. The one-letter and three-letter abbreviations for natural amino acids are listed below.

[0021] "Factor Xa" or "fXa" or "fXa protein" is a serine protease in the blood coagulation pathway that is produced from inactive factor X (fX, SEQ ID NO: 1, Table 1). The nucleotide sequence encoding human factor X ("fX") can be found in GenBank under the accession number "NM_000504." fX is activated to fXa by catalytic cleavage of the first 52 residues of the heavy chain. FXa comprises a light chain and a heavy chain. The first 45 amino acid residues of the light chain (residues 1-45 of SEQ ID NO: 1) contain 11 post-translationally modified gamma-carboxyglutamic acid (Gla) residues and are therefore referred to as the Gla domain. It also contains a short (6 amino acid residue) aromatic stack sequence (residues 40-45 of SEQ ID NO: 1). Chymotrypsin digestion selectively removes residues 1-44, resulting in fXa lacking the Gla domain. The serine protease catalytic domain of fXa is located in the C-terminal heavy chain, which is highly homologous to other serine proteases such as thrombin, trypsin, and activated protein C.

[0022] "Native fXa" or "wild-type fXa" refers to fXa that is naturally occurring in plasma or isolated in its original, unmodified form, which possesses the biological activity of activating prothrombin and thus promoting the formation of a blood clot. This term includes naturally occurring polypeptides isolated from tissue samples and recombinantly produced fXa. "Active fXa" refers to fXa that has procoagulant activity to activate prothrombin. "Active fXa" can be native fXa or modified fXa that retains procoagulant activity.

[0023] As used herein, "fXa antidote," "antidote," or "fXa derivative" refers to a modified fXa protein that does not compete with fXa for incorporation into the prothrombinase complex, has reduced or no procoagulant or catalytic activity, and further binds to and / or substantially neutralizes anticoagulants, such as fXa inhibitors. In some embodiments, the "procoagulant activity" of a fXa protein or fXa derivative refers to the enzymatic activity retained by a wild-type active fXa polypeptide. Examples of fXa derivatives are provided in U.S. Pat. No. 8,153,590 and PCT Publications WO 2009 / 042962 and WO 2010 / 056765, and are further provided herein, such as SEQ ID NOS: 2 and 3 and their biological equivalents.

[0024] "Enzymatic activity" of a fXa polypeptide or derivative thereof refers to the ability of the polypeptide to catalyze a biochemical reaction with a substrate through direct interaction with the substrate.

[0025] SEQ ID NO:2 contains three mutations compared to wild-type fXa. The first mutation is a deletion of aa 6-39 in the Gla domain of fX. The second mutation is a substitution of aa 143-194 of the activation peptide sequence with -RKR-. This generates a -RKRRKR- (SEQ ID NO:6) linker connecting the light chain (SEQ ID NO:4) and heavy chain (SEQ ID NO:5). Upon secretion, this linker is cleaved, resulting in the dual-chain polypeptide, SEQ ID NO:3 (r-Antidote). The third mutation is a mutation of the active site residue S379 to an Ala residue. This amino acid substitution corresponds to amino acids 296 and 290 in SEQ ID NOs:1 and 3, respectively.

[0026] An example of an antidote is "r-Antidote," which refers to the processed double-chain polypeptide product of SEQ ID NO: 2 after linker cleavage. This is represented by SEQ ID NO: 3. r-Antidote is disclosed, for example, in US Pat. No. 8,153,590, the contents of which are incorporated herein by reference. r-Antidote comprises a light chain (SEQ ID NO: 4) and a heavy chain (SEQ ID NO: 5) linked by a single disulfide bond between cysteine ​​98 (Cys98) of the light chain and cysteine ​​108 (Cys108) of the heavy chain. Similar to wild-type fXa, in certain production batches, r-Antidote undergoes post-translational modifications, such as glycosylation at specific amino acid residues, such as Ser56, Ser72, Ser76, and Thr82 of the light chain and Thr249 of the heavy chain, as well as modified residues, such as (3R)-3-hydroxyAsp at Asp29 of the light chain. Furthermore, in addition to the interchain disulfide bonds, there may be intrachain disulfide bonds formed between cysteines 16 and 27, 21 and 36, 38 and 47, 55 and 66, 62 and 75, 77 and 90 of the light chain and between cysteines 7 and 12, 27 and 43, 156 and 170, and 181 and 209 of the heavy chain. [Table 1] [Table 2] [Table 3] [Table 4]

[0027] Other examples of antidotes can be biological equivalents of r-Antidote (or their precursors as shown in SEQ ID NO: 2) or polypeptides having specific sequence identity to SEQ ID NO: 3. In one embodiment, such biological equivalents retain the structural properties of SEQ ID NO: 3, i.e., a modified active site and a deleted or modified Gla domain. In another embodiment, such biological equivalents retain the functional properties of SEQ ID NO: 3, i.e., no competition with fXa for incorporation into the prothrombinase complex and reduced or no procoagulant (e.g., enzymatic or catalytic) activity.

[0028] The term "active site" refers to the portion of an enzyme or antibody where a chemical reaction occurs. An "altered active site" is a site that has been structurally altered to result in an active site with increased or decreased chemical reactivity or specificity. Examples of active sites include, but are not limited to, the catalytic domain of human factor X comprising amino acid residues 235-488 and the catalytic domain of human factor Xa comprising amino acid residues 195-448 of SEQ ID NO: 1, with at least one amino acid substitution at positions Arg306, Glu310, Arg347, Lys351, Lys414, or Arg424.

[0029] Preparation of antidote The experimental examples (Examples 1 and 2) demonstrate the development of culture and purification methods for preparing an fXa antidote. The cultured cells contain a polynucleotide construct comprising a nucleic acid sequence encoding the nucleic acid sequence of SEQ ID NO:7 or an amino acid sequence having at least 90% sequence identity (or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence encoded by SEQ ID NO:7. The cells are typically Chinese hamster ovary (CHO) cells.

[0030] One purification method is illustrated in Figure 1. After cells are harvested (step 101) and clarified using a depth filter to remove high molecular weight (HWM) impurities, the sample is concentrated (e.g., approximately 10-fold) (step 102). The concentration step is not limited and can use regenerated cellulose. In step 103, viral inactivation (e.g., using a detergent / solvent such as 1% Triton X-100 and 0.3% tributyl phosphate (final concentration)) can be performed to inactivate enveloped viruses in cell culture. Following viral removal, steps 104 (mixed-mode cation exchange), 105 (mixed-mode anion exchange), and 106 (mixed-mode ion exchange) are performed to remove host cell proteins and DNA and capture antidote agents. In step 107, a hydrophobic interaction resin can be used to further remove remaining host cell proteins. Optionally, these purification steps can be followed by a final viral removal filtration step to remove any remaining viruses.

[0031] In another embodiment, the purification step is illustrated in FIG. 2 and demonstrated in Example 2. In some embodiments, the method includes adding a detergent to a sample containing a polynucleotide construct (e.g., SEQ ID NO: 7) and a polypeptide product (e.g., SEQ ID NO: 3) expressed from the polynucleotide construct. In some embodiments, the detergent includes Triton X-100. In some embodiments, no solvent is used to treat the sample before it is subjected to subsequent affinity purification. In some embodiments, no organic solvent is used to treat the sample before it is subjected to subsequent affinity purification. In some embodiments, tributyl phosphate is not added to the sample before it is subjected to subsequent affinity purification.

[0032] In some embodiments, the sample is then loaded onto a soybean trypsin inhibitor (STI)-based affinity chromatograph and eluted with an elution buffer to produce an eluted sample, hi some embodiments, the loaded sample is not treated with or does not contain an organic solvent.

[0033] "STI" or "soybean trypsin inhibitor" refers to a trypsin inhibitor isolated from soybeans or their biological equivalents. The trypsin inhibitor is approximately 20 kDa in size and reduces trypsin (a proteolytic enzyme) and plasma kallikrein, factor Xa, and plasmin activity. STIs are commercially available from suppliers such as Life Technologies (Grand Island, NY). An example of an STI is KTI3 Kunitz trypsin inhibitor from soybean (Glycine max) having GenBank accession number NP_001238611.

[0034] For the purification of specific proteins, STIs can be immobilized on solid support resins. In addition to soybean trypsin inhibitor, other trypsin inhibitor proteins, such as those isolated from serum, lima bean, bovine pancreas, or ovomucoid, or modified forms thereof, can also be used. It is also contemplated that specific protease inhibitors, particularly serine protease inhibitors, can be used to prepare affinity resins for the purpose of purifying antidotes. The antidote can then be eluted with a buffer containing arginine. In some embodiments, the elution buffer contains at least 0.2M, 0.5M, 0.6M, 0.7M, 0.8M, 0.9M, 1M, 1.1M, 1.2M, 1.3M, 1.4M, 1.5M, or 2M arginine. In some embodiments, the elution buffer comprises about 0.5 M to 2 M arginine, or about 0.7 M to 1.5 M arginine, or 0.8 M to 1.2 M arginine. In some embodiments, the elution buffer has a pH of about 4.5 to 6, or 4.6 to 5.6, or 4.7 to 5.5, or 4.8 to 5.4, or 5 to 5.4, or 5.1 to 5.3, or about 5.2. In one embodiment, the elution buffer comprises 25 mM sodium acetate, 1.0 M arginine at pH 5.2.

[0035] In some embodiments, the sample is then loaded onto an ion exchange and mixed-mode chromatograph. Non-limiting examples of ion exchange and mixed-mode chromatographs include ceramic hydroxyapatite Type I chromatographs. The sample may then be eluted with an elution buffer containing an inorganic salt. In some embodiments, the inorganic salt is sodium chloride or potassium chloride. In some embodiments, the concentration of the salt in the elution buffer is at least 0.5M, 1M, 1.5M, 2M, 2.5M, or 3M.

[0036] In some embodiments, elution involves the use of a gradient developed between an equilibration buffer and an elution buffer. The equilibration buffer, in some embodiments, contains a lower concentration of salt, e.g., less than 0.2 M, less than 0.1 M, less than 50 mM, less than 20 mM, or less than 10 mM. In some embodiments, the gradient comprises at least a 5-fold, 10-fold, 20-fold, 50-fold, or 100-fold increase in salt concentration. In some embodiments, the equilibration buffer contains 50 mM MES (2-(N-morpholino)ethanesulfonic acid), 5 mM sodium phosphate at pH 7.0. In some embodiments, the elution buffer contains 50 mM MES, 5 mM sodium phosphate, 2 M sodium chloride at pH 7.0. In some embodiments, the gradient begins at about 90% equilibration buffer and ends at about 90% elution buffer.

[0037] In some embodiments, the sample is further loaded onto a hydrophobic interaction chromatograph. In some embodiments, the hydrophobic interaction chromatograph comprises an octyl sepharose chromatograph. In some embodiments, the sample is eluted with an elution buffer comprising at least 2 mM sodium chloride, or at least 1 M, 1.5 M, 2.5 M, 3 M, 4 M, or 5 M sodium chloride.

[0038] In some embodiments, one of the intermediate samples described above is subjected to a further purification step with anion exchange chromatography. In some embodiments, the anion exchange chromatography comprises a Sartobind™ ion exchange membrane. In some embodiments, the anion exchange chromatography is applied after the affinity chromatography.

[0039] In some embodiments, one of the intermediate samples described above is further subjected to filtration through a nanofleece filter, which in some embodiments is applied before loading the sample onto an STI-based affinity chromatograph.

[0040] In some embodiments, the purified sample contains less than about 1% contaminating host cell proteins or proteins not expressed by the polynucleotide construct, such as STI, that have been released from the affinity resin / column.

[0041] The purification process of any of the above embodiments may recover at least about 50% (or at least about 40%, 45%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%) of the antidote protein expressed in the cell culture. In some embodiments, the cells are grown in culture under conditions to produce at least 100 mg of polypeptide product per liter of culture. In some embodiments, the cells are grown in culture under conditions to produce at least 120 mg (or at least 210 mg, 220 mg, 230 mg, 240 mg, 250 mg, 270 mg, 280 mg, 290 mg, 300 mg, 310 mg, 320 mg, 330 mg, 340 mg, or 350 mg) of polypeptide product per liter of culture. In some embodiments, purified samples contain more than about 50 mg (or at least 55 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg, 160 mg, 170 mg, 180 mg, 190 mg, or 200 mg) of polypeptide product produced per liter of culture.

[0042] Antidote isomers in purified products The purified antidote product from any of the methods of the present disclosure is expected to contain light and heavy chains, however, the cultivation and purification process may introduce changes into the actual protein.

[0043] As demonstrated in Example 3, approximately 20% to 50% of the polypeptide products in the purified sample have an intact heavy chain (SEQ ID NO: 5). Of those with intact heavy chains, approximately 5% to 95% of the heavy chains have two O-linked glycosylations, and approximately 5% to 95% of the heavy chains have one O-linked glycosylation.

[0044] In some embodiments, at least about 20%, 25%, 30%, 35%, 40%, or 45% of the polypeptide products in a purified sample have intact heavy chains, hi some embodiments, no more than about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% of the polypeptide products in a purified sample have intact heavy chains.

[0045] In some embodiments, about 40% to 80% of the polypeptide products in a purified sample have a heavy chain lacking a C-terminal lysine (SEQ ID NO: 8). In some embodiments, SEQ ID NO: 8 comprises at least about 20%, 25%, 30%, 35%, 40%, 45%, or 50% of the purified sample. In some embodiments, SEQ ID NO: 8 comprises no more than about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the purified sample. In some embodiments, at least about 90% of the heavy chains of SEQ ID NO: 8 have one O-linked glycosylation.

[0046] In some embodiments, about 2% to 12% of the polypeptide products in a purified sample have a heavy chain lacking 13 C-terminal amino acid residues (SEQ ID NO: 9). In some embodiments, SEQ ID NO: 9 constitutes at least about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, or 5% of the purified sample. In some embodiments, SEQ ID NO: 9 constitutes no more than about 2%, 3%, 4%, 5%, 7%, 10%, 15%, 17%, or 20% of the purified sample.

[0047] In some embodiments, about 0.1% to 1.5% of the polypeptide products in a purified sample have a heavy chain lacking 14 C-terminal amino acid residues (SEQ ID NO: 10). In some embodiments, SEQ ID NO: 10 comprises at least about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, or 0.5% of the purified sample. In some embodiments, SEQ ID NO: 10 comprises no more than about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, or 3% of the purified sample.

[0048] In some embodiments, about 2% to 8% of the polypeptide products in a purified sample have a heavy chain lacking 15 C-terminal amino acid residues (SEQ ID NO: 11). In some embodiments, SEQ ID NO: 11 comprises at least about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, or 5% of the purified sample. In some embodiments, SEQ ID NO: 11 comprises no more than about 5%, 6%, 7%, 8%, 9%, or 10% of the purified sample.

[0049] Example 3 also shows that approximately 35% to 60% of the polypeptide products in a purified sample have an intact light chain (SEQ ID NO: 4), while some others may be modified or truncated. In some embodiments, the amount of intact light chains in the total number of light chains is at least about 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, the amount of intact light chains in the total number of light chains is no more than about 45%, 50%, 55%, 60%, 65%, 70%, 80%, or 90%.

[0050] In some embodiments, the disclosure provides a pharmaceutical product comprising a pharmaceutically acceptable carrier and a polypeptide portion of a double-chain polypeptide, wherein approximately 35%-60% of the double-chain polypeptides have light chains consisting of the amino acid sequence of SEQ ID NO: 4. In some embodiments, approximately 20%-50% of the double-chain polypeptides have heavy chains consisting of the amino acid sequence of SEQ ID NO: 5. In some embodiments, approximately 40%-80% of the double-chain polypeptides have heavy chains consisting of the amino acid sequence of SEQ ID NO: 8. In some embodiments, less than approximately 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3% of the double-chain polypeptides have heavy chains consisting of the amino acid sequence of SEQ ID NO: 9. In some embodiments, approximately 0.1%-1.5% of the double-chain polypeptides have heavy chains consisting of the amino acid sequence of SEQ ID NO: 10. In some embodiments, approximately 2%-8% of the double-chain polypeptides have heavy chains consisting of the amino acid sequence of SEQ ID NO: 11.

[0051] In some embodiments, about 5% to 95% of the heavy chains consisting of the amino acid sequence of SEQ ID NO: 5 have two O-linked glycosylations. In some embodiments, about 5% to 95% of the heavy chains consisting of the amino acid sequence of SEQ ID NO: 5 have one O-linked glycosylation. In some embodiments, at least about 90% of the heavy chains consisting of the amino acid sequence of SEQ ID NO: 8 have one O-linked glycosylation.

[0052] Formulations, Methods and Dosages Formulations prepared with purified antidote protein product are also provided. In some embodiments, aqueous formulations suitable for lyophilization are provided. In one embodiment, the formulation comprises an antidote along with a solubilizer, a stabilizer (or stabilizers), and a crystalline agent. The formulation may further comprise a surfactant and / or a buffer. In some aspects, the presence of each of these agents prevents degradation of the antidote during lyophilization, for example, at lyophilization temperatures greater than -40°C, -30°C, -20°C, -10°C, 0°C, 5°C, 10°C, or 15°C, and as high as 20°C or 25°C.

[0053] "Crystalline components" refer to molecules that form a crystalline matrix during the lyophilization process in a formulation containing a polypeptide. Non-limiting examples of crystalline components include mannitol and glycine.

[0054] In some embodiments, the crystalline component is mannitol (e.g., crystalline mannitol). In one embodiment, the concentration of the crystalline component in the aqueous formulation is at least 1% (w / v). In one embodiment, the concentration of the crystalline component in the aqueous formulation is at least 1.5%, 2%, 2.5%, 3%, 3.5%, or 4% (w / v). In one embodiment, the concentration of the crystalline component in the aqueous formulation is no greater than 8%, or no greater than 7%, 6.5%, 6%, 5.5%, 5%, 4.5%, or 4% (w / v). In one aspect, the concentration of the crystalline component in the aqueous formulation is about 1% to about 8%, or about 2% to about 6%, or about 3% to about 5.5%, or about 4.5% to about 5.5%, or about 4.6% to about 5.4%, or about 4.7% to about 5.3%, or about 4.8% to about 5.2%, or about 4.9% to about 5.1%, or about 4%, 4.5%, or 5% (w / v).

[0055] In some embodiments, a solubilizing agent is included in the aqueous formulation. The term "solubilizing agent" refers to salts, ions, carbohydrates, complexing agents, polymers, and other compounds that, when present in a solution, increase the solubility of another molecule (e.g., an active ingredient) in the solution. Non-limiting examples of solubilizing agents include arginine and citric acid. In one embodiment, the solubilizing agent is arginine. In one embodiment, the solubilizing agent is citric acid.

[0056] The presence of a solubilizing agent can be useful to keep the fXa polypeptide soluble and stable in the formulation. In some embodiments, the concentration of the solubilizing agent (e.g., arginine) is at least 10 mM, or at least 20 mM, 25 mM, 30 mM, 36 mM, or 40 mM. In some embodiments, the concentration of the solubilizing agent (e.g., arginine) is no greater than 100 mM, 96 mM, 90 mM, 80 mM, 70 mM, 60 mM, or 50 mM. In some embodiments, the concentration of the solubilizing agent is about 10 mM or 20 mM to about 60 mM, about 10 mM or 20 mM to about 55 mM, about 35 mM to about 55 mM, about 40 mM to about 50 mM, about 41 mM to about 49 mM, about 42 mM to about 48 mM, about 43 mM to about 47 mM, about 44 mM to about 46 mM, or 40 mM, 45 mM, or 50 mM. Note that the term arginine, as used herein, refers to amino acids and salts thereof (e.g., arginine HCl). Arginine has a molecular weight of about 174.2 daltons, and arginine HCl (e.g., L-arginine HCl, L-arginine acetate) has a molecular weight of about 210.7 daltons.

[0057] In one embodiment, the solubilizing agent is citric acid or a salt thereof. The citric acid salt is sodium citrate. In one aspect, the citric acid comprises a concentration of about 1.0 mM to about 200.0 mM. In a further aspect, the citric acid comprises a concentration of about 25 mM. In another aspect, the citric acid comprises a concentration of about 50 mM. In a further embodiment, the citric acid comprises a concentration of about 5 mM, 10 mM, or 20 mM. In another embodiment, the citric acid comprises a concentration of about 0.05 mM to about 0.2 mM.

[0058] In some embodiments, a stabilizer is included in the aqueous formulation. The term "stabilizer" refers to a pharmaceutically acceptable excipient that protects the active ingredient (e.g., fXa derivative polypeptide) and / or formulation from chemical and / or physical degradation during manufacturing, storage, and application. Examples of stabilizers include sucrose, arginine, citric acid, mannitol, trehalose, glycine, sodium chloride, dextran, and glucose. In one embodiment, the stabilizer is sucrose.

[0059] In one embodiment, the concentration of the stabilizer (e.g., sucrose) in the aqueous formulation is at least about 0.5% (w / v). In one embodiment, the concentration of the stabilizer (e.g., sucrose) in the aqueous formulation is at least about 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2% (w / v). In one embodiment, the concentration of the stabilizer (e.g., sucrose) in the aqueous formulation does not exceed about 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, or 2% (w / v). In one aspect, the concentration of the stabilizer (e.g., sucrose) in the aqueous formulation is about 1% to about 5%, or about 1% to about 4%, or about 1% to about 3%, or about 1.5% to about 2.5%, or about 1.6% to about 2.4%, or about 1.7% to about 2.3%, or about 1.7% to about 2.2%, or about 1.9% to about 2.1%, or about 1%, 1.5%, 2%, 2.5%, or 3% (w / v).

[0060] In some embodiments, the aqueous formulation can further comprise a surfactant, a buffering agent, an isotonicity agent, a cryoprotectant, a surfactant, a cryoprotectant, a preservative, or a combination thereof.

[0061] In some embodiments, the aqueous formulation has a pH of 6 or greater, or 6.5 or greater, or 7 or greater, or 7.5 or greater. In some embodiments, the pH is no higher than 9, 8.5, or 8. In some embodiments, the pH is 6-9, 6.5-8.5, 7-8.5, 7.5-8.2, 7.6-8.1, 7.7-7.9, or about 7.5, 7.6, 7.7, 7.8, 7.9, or 8.

[0062] In one embodiment, the aqueous formulation comprises about 45 mM arginine, about 2% sucrose (w / v), about 5% mannitol (w / v), and about 10 mg / mL double-chain r-Antidote, and the formulation has a pH of about 7.8. In one embodiment, the aqueous formulation comprises about 45 mM arginine, about 2% sucrose (w / v), about 5% mannitol (w / v), and about 20 mg / mL double-chain r-Antidote, and the formulation has a pH of about 7.8. In one embodiment, the aqueous formulation comprises about 45 mM arginine, about 2% sucrose (w / v), about 5% mannitol (w / v), and about 40 mg / mL double-chain r-Antidote, and the formulation has a pH of about 7.8. In one embodiment, the aqueous formulation further comprises 0.01%-0.02% (w / v) polysorbate 80 and a buffer.

[0063] In some embodiments, lyophilized compositions prepared by lyophilizing the aqueous formulations of the present disclosure are also provided. Based on the concentration of each agent in the aqueous formulation, the relative content of the agents in the lyophilized composition can be readily determined.

[0064] In one embodiment, the lyophilized composition comprises at least 5%, alternatively at least 10%, 15%, 20%, 25%, 30%, or 35% (w / w) of fXa antidote, and then, among other main components, for example, the weight ratio of L-arginine HCl:sucrose:mannitol can be in the range of (0.5-1.4):(1-3):(2-6). In some embodiments, the weight ratio of L-arginine HCl:sucrose:mannitol is in the range of (0.9-1):(1.5-2.5):(4.5-5.5), or (0.91-0.99):(1.6-2.4):(4.6-5.4), or (0.92-0.98):(1.7-2.3):(4.7-5.3), (0.93-0.97):(1.8-2.2):(4.8-5.2), or (0.94-0.96):(1.9-2.1):(4.9-5.1). In some embodiments, the lyophilized composition further comprises a solid portion of a surfactant and / or buffer.

[0065] The present disclosure also relates to a method of treating, preventing, or reducing bleeding in a subject undergoing anticoagulant therapy with an fXa inhibitor, comprising administering to the subject an effective amount of the lyophilized formulation dissolved in a suitable solvent. It is contemplated that the disclosed antidotes or derivatives may be short-term agents used in elective or emergency situations, and can safely and specifically neutralize the traditional anticoagulant properties of fXa inhibitors without causing adverse hemodynamic side effects or exacerbating the proliferative vascular response to injury.

[0066] As used herein, the terms "treating," "treatment," and the like are used herein to mean obtaining a desired pharmacological and / or physiological effect. That effect may be prophylactic, in terms of completely or partially preventing a disease or its signs or symptoms, and / or therapeutic, in terms of partially or completely curing a disease and / or adverse effects resulting from a disease.

[0067] "Treating" also encompasses any treatment of a disease in a mammal, including (A) preventing the disease from occurring in a subject who may be predisposed to the disease but who may not yet be diagnosed as having the disease (e.g., preventing bleeding in a patient with an anticoagulant overdose), (b) inhibiting the disease (i.e., preventing its occurrence, e.g., suppressing bleeding), or (c) alleviating or ameliorating the disease (e.g., reducing bleeding).

[0068] As used herein, "treating" further includes the general amelioration of symptoms associated with a medical condition and / or delay in the onset of symptoms. Clinical and subclinical evidence of "treatment" varies depending on the pathology, the individual, and the treatment.

[0069] "Administration" can be a single administration, continuous administration, or intermittent administration throughout the course of treatment. Methods for determining the most effective means and dosage of administration are known to those skilled in the art and vary with the composition used for treatment, the purpose of the treatment, the target cell being treated, and the subject being treated. Single or multiple administrations can be used, with the dosage level and pattern selected by the treating physician. Suitable dosage formulations and methods for administering agents are known in the art. A "subject" for diagnosis or treatment can be a cell or a mammal, including a human. Non-human animal subjects for diagnosis or treatment include, for example, murines, such as rats and mice, canines, such as dogs, lagomorphs, such as rabbits, farm animals, sport animals, and pets.

[0070] The medicaments and compositions of the disclosure can be used in the manufacture of medicaments and for the treatment of humans and other animals by administration in accordance with conventional procedures as the active ingredient in a pharmaceutical composition.

[0071] The agents of the present disclosure can be administered for therapy by any suitable route, particularly parenteral (including subcutaneous, intramuscular, intravenous, and intradermal) administration, it being understood that the preferred route will vary with the condition and age of the recipient, and the disease being treated.

[0072] The expression "pharmaceutically acceptable polymer" refers to a group of compounds that can be attached to one or more of the polypeptides described herein. It is contemplated that attachment of a polymer to a polypeptide can extend the half-life of the polypeptide in vivo and in vitro. Non-limiting examples include polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, cellulose derivatives, polyacrylates, polymethacrylates, sugars, polyols, and mixtures thereof.

[0073] An "anticoagulant" or "anticoagulant" is a drug that inhibits blood clot formation. Examples of anticoagulants include, but are not limited to, thrombin, inhibitors specific for factor IXa, Xa, XIa, XIIa, or VIIa, heparin and derivatives, vitamin K antagonists, and anti-tissue factor antibodies. Examples of specific inhibitors of thrombin include hirudin, bivalirudin (Angiomax®), argatroban, and lepirudin (Refludan®). Examples of heparins and derivatives include unfractionated heparin (UFH), low molecular weight heparins (LMWH) such as enoxaparin (Lovenox®), dalteparin (Fragmin®, and danaparoid (Orgaran®), and synthetic pentasaccharides such as fondaparinux (Arixtra®). Examples of vitamin K antagonists include warfarin (Coumadin®), phenocumarol, acenocoumarol (Sintrom®), chlorindione, dicumarol, diphenadione, ethyl viscoum acetate, phenprocoumon, phenindione, and thiochromarol. In one embodiment, the anticoagulant is an inhibitor of factor Xa. In one embodiment, the anticoagulant is betrixaban.

[0074] "Anticoagulant therapy" refers to a therapy administered to a patient to prevent unwanted blood clots or thrombosis. Anticoagulant therapy involves the administration of one or a combination of two or more anticoagulants or other agents at doses and schedules suitable to treat or prevent unwanted blood clots or thrombosis in a patient.

[0075] The terms "factor Xa inhibitor" or "inhibitor of factor Xa" refer to compounds that are capable of directly or indirectly inhibiting the activity of coagulation factor Xa, which catalyzes the conversion of prothrombin to thrombin in vitro and / or in vivo.

[0076] "Direct factor Xa inhibitors" bind directly to fXa, and non-limiting examples include NAP-5, rNAPc2, tissue factor pathway inhibitor (TFPI), DX-DX-9065a (described, for example, in Herbert, JM, et al, J Pharmacol Exp Ther. 1996 276(3):1030-8), YM-60828 (described, for example, in Taniuchi, Y., et al, Thromb Haemost. 1998 79(3):543-8), YM-150 (described, for example, in Eriksson, B.I. et al, Blood 2005;106(11), Abstract 1865), apixaban, rivaroxaban, TAK-442, PD-348292 (described, for example, in Pipeline Insight: Antithrombotics—Reaching the Untreated Prophylaxis Market, 2007), otamixaban, edoxaban (e.g., Hylek EM, Curr Opin Invest Drugs 2007 8(9):778-783), LY517717 (e.g., as described in Agnelli, G., et al, J. Thromb. Haemost. 2007 5(4):746-53), GSK913893, razaxaban, betrixaban, or pharmaceutically acceptable salts thereof, and combinations thereof. In certain embodiments, the direct factor Xa inhibitor is rivaroxaban. In some embodiments, the direct factor Xa inhibitor is a small molecule chemical compound.

[0077] The inhibition of fXa activity of an "indirect factor Xa inhibitor" is mediated by one or more other factors. Non-limiting examples of indirect factor Xa inhibitors include fondaparinux, idraparinux, biotinylated idraparinux, enoxaparin, fragmin, tinzaparin, low molecular weight heparin ("LMWH"), and combinations thereof. In a particular embodiment, the indirect factor Xa inhibitor is enoxaparin.

[0078] In one embodiment, the factor Xa inhibitor is selected from betrixaban, rivaroxaban, LMWH, DX-9065a, YM-60828, YM-150, PD-348292, otamixaban, edoxaban, LY517717, GSK913893, razaxaban, apixaban, and combinations thereof.

[0079] The term "betrixaban" refers to the compound "[2-({4-[(dimethylamino)iminomethyl]phenyl}carbonylamino)-5-methoxyphenyl]-N-(5-chloro(2-pyridyl))carboxamide" or a pharmaceutically acceptable salt thereof. Betrixaban is described in U.S. Pat. Nos. 6,376,515, 6,835,739, and 7,598,276, the contents of which are incorporated herein by reference. Betrixaban is known as a specific inhibitor of factor Xa.

[0080] "Neutralizing," "antagonizing," or "counteracting" the activity of an inhibitor of fXa, or similar expressions, refers to inhibiting or preventing the factor Xa inhibitory or anticoagulant function of an fXa inhibitor. Such expressions refer to partial inhibition or prevention of function, as well as inhibition or prevention of most or all of the fXa inhibitor activity, in vitro and / or in vivo.

[0081] An "effective amount" refers to the amount of a derivative sufficient to produce a desired biological and / or therapeutic result. The result may be the alleviation of signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In the present disclosure, the result typically includes one or more of the following: neutralization of the fXa inhibitor administered to the patient, reversal of the anticoagulant activity of the fXa inhibitor, removal of the fXa inhibitor from the plasma, restoration of hemostasis, and reduction or cessation of bleeding. The effective amount will vary depending on the particular antidote used, the particular fXa inhibitor administered to the subject, the dosing regimen of the fXa inhibitor, the timing of administration of the antidote, the subject and disease state being treated, the weight and age of the subject, the severity of the disease state, the mode of administration, etc., all of which can be readily determined by one of ordinary skill in the art.

[0082] In certain embodiments, the solution is administered to deliver an amount of fXa antidote ranging from about 10 milligrams (mg) to about 2 grams (g). Other amounts of r-antidote used include about 100 mg to about 1.5 g, about 200 mg to about 1 g, and about 400 mg to about 900 mg. In some embodiments, the amount of r-antidote used is about 400 mg or 960 mg. In some embodiments, the amount of r-antidote used is about 10 mg to about 100 mg, about 15 mg to about 95 mg, or about 20 mg to about 80 mg.

[0083] Administration of the formulation neutralizes the factor Xa inhibitor by at least about 20%, or at least about 50%, or at least about 75%, or at least about 90%, or at least about 95%.

[0084] A number of in vitro assays, such as thrombin generation assays, and clinical clotting assays, such as aPTT, PT, and ACT, can determine whether this method, i.e., inhibition or antagonism of factor Xa inhibitors, is achieved.

[0085] One aspect of the present disclosure relates to a method for selectively binding to and inhibiting an exogenously administered fXa inhibitor in a subject undergoing anticoagulant therapy with an fXa inhibitor, comprising administering to the subject an effective amount of a solution of a lyophilized formulation. Patients suitable for this therapy are undergoing conventional anticoagulant therapy, e.g., receiving one or more anticoagulants (e.g., direct or indirect inhibitors of fXa).

[0086] In another aspect, a method provided herein selectively binds to and inhibits exogenously administered factor Xa inhibitors in a subject undergoing anticoagulant therapy with a factor Xa inhibitor, comprising administering to the subject a solution of a lyophilized formulation. The subject may be a cell or a mammal, such as a human.

[0087] Subjects who may benefit from the administration of the reconstituted lyophilized formulations and associated methods described herein include those experiencing or predisposed to clinically significant or clinically significant non-massive bleeding events. Examples of clinically significant bleeding events are selected from the group consisting of hemorrhage, bleeding into vital organs, bleeding requiring reoperation or a new therapeutic procedure, and a bleeding index of 2.0 or greater with associated overt bleeding (Turpie AGG, et al., NEJM, 2001, 344:619-625). Additionally, subjects may experience or be predisposed to non-massive bleeding events selected from the group consisting of persistent or recurrent epistaxis that may be substantial or may not stop without intervention, rectal or urinary tract bleeding that does not rise to a level requiring a therapeutic procedure, substantial hematoma at the injection site or elsewhere that may be spontaneous or occur with minor trauma, significant blood loss beyond that typically associated with surgical procedures that does not require drainage, and bleeding requiring unplanned blood transfusion.

[0088] In some embodiments, the reconstituted lyophilized formulation is administered after an overdose of an fXa inhibitor or prior to surgery that may place the subject at risk for bleeding. [Example]

[0089] The following examples are included to demonstrate specific embodiments of the present disclosure. It will be understood by those skilled in the art that the techniques disclosed in the examples below represent techniques that work well in the practice of the present disclosure and, therefore, can be considered to constitute specific modes for its practice. However, in light of the present disclosure, those skilled in the art should recognize that many changes can be made in the specific embodiments disclosed and still obtain like or similar results without departing from the spirit and scope of the present disclosure.

[0090] Example 1: Purification of r-Antidote from Cell Culture This example demonstrates the development of culture conditions and a subsequent purification process for the production of r-Antidote. Commercially available culture media compatible with CHO cells were tested on CHO cells transfected with a construct containing a nucleic acid sequence (SEQ ID NO: 7) encoding the r-Antidote precursor (SEQ ID NO: 2) that generates duplex r-Antidote (SEQ ID NO: 3) upon removal of the -RKRRKR- linker (SEQ ID NO: 6). ProCHO™ medium, under suitable conditions, was capable of producing titers as high as 75-95 mg / L.

[0091] A subsequent purification process ("Subsequent Process") was developed to purify the antidote from the cultured cells. In several early development runs, various membranes and columns were tested.

[0092] In two test runs, the clarified harvest was found to either foul the membrane when the product was concentrated by ultrafiltration (UF) or to foul the chromatography media when UF was avoided and the product was concentrated by a first chromatography step. Because product concentration was deemed necessary early in the experiment, experiments were conducted to identify the cause of the fouling and determine steps to remove it. It was subsequently determined that the fouling was caused by a high-molecular-weight (HMW) protein impurity secreted into the medium during cell culture. This HMW protein was only partially soluble in the clarified harvest and became further insoluble upon concentration, causing unacceptable fouling of the UF membrane or chromatography column.

[0093] After evaluation of several depth filters, ion exchange membranes, and chromatography media, it was found that one particular depth filter (Millipore™ X0HC grade filter) was capable of removing the majority of impurities while achieving acceptable product yield. The product was then concentrated up to 10-fold by UF. This filter replaced one of the original filters in the clarification process and was implemented in two additional runs.

[0094] Figure 1 shows the purification process 100 from this development effort. After cells are harvested (step 101) and clarified using a Millipore™ X0HC filter to remove HWM impurities, the sample is concentrated 10-fold (step 102). Regenerated cellulose can be used for concentration. In step 103, viral inactivation (using a detergent / solvent such as 1% Triton X-100 with a final concentration of 0.3% tributyl phosphate) can be performed to inactivate enveloped viruses in the cell culture. Following viral removal, steps 104 (mixed-mode cation exchange), 105 (mixed-mode anion exchange), and 106 (mixed-mode ion exchange) can be performed to remove host cell proteins and DNA and capture antidote agents. In step 107, a hydrophobic interaction resin can be used to further remove remaining host cell proteins. Optionally, these purification steps can be followed by a final viral removal filtration step to remove any remaining viruses.

[0095] This process was able to recover approximately 30–35% of the antidote expressed in the cell culture medium, producing approximately 22–33 mg of antidote protein per liter of cell culture medium.

[0096] Example 2: Scale-up manufacturing process Based on the process shown in Example 1, an improved process was developed for the purpose of scaling up production volumes.

[0097] The culture medium of Example 1 was able to produce a titer of approximately 75-95 mg / L. To increase production, several other CHO culture media were tested. One of them showed a 2-3 fold increase in cell growth and peak cell density (titer = 200-225 mg / L), allowing for scale-up to 10,000 L.

[0098] An affinity chromatograph was developed to extract antidotes in large quantities. The affinity ligand included Kunitz trypsin inhibitor (21 kDa; pI = 4.5) extracted from whole soybeans or wheat flour. Soybean trypsin inhibitor (STI) forms a 1:1 complex with the antidote. For scalability and compatibility, elution conditions for the noncompetitive inhibitor were identified and included 25 mM sodium acetate, 1 M arginine at pH 5.2.

[0099] In the process developed in Example 1, a depth filter harvest clarification step was used to remove HMW impurities, followed by a 10-fold concentration. Additionally, a viral inactivation step utilized a detergent (Triton X-100) and a solvent (tributyl phosphate). The suitability and efficiency of these steps were evaluated with respect to affinity capture.

[0100] This data indicates that depth filtration is not necessary and that the use of solvents and a 10-fold concentration step reduces product recovery. Therefore, in further development, the depth filtration and concentration steps were eliminated, and viral inactivation was performed using only detergent (Triton X-100) without solvents. Viral inactivation was achieved by adding 10% Triton X-100 at a ratio of 52.7 ml of buffer per liter of product (final concentration: 0.5% Triton X-100). This finding was unexpected, as solvents are typically used for sample preparation before loading onto the affinity column. Also, note that when the cell culture was harvested, a flow-through centrifugation step was used to remove precipitates.

[0101] After a 15 minute mixing time at room temperature and a minimum 1 hour hold period, the processed product was filtered through a filter train consisting of a Sartogruad NF filter (0.8 / 0.2 μm) prefilter to a Sartopore 2 (0.45 / 0.22 μm) prefilter, followed by a 500 L vessel containing 0.22 μm filters. The charge of each column was filtered on the day of use.

[0102] Elution of the product from the STI affinity resin used an elution buffer containing arginine (25 mM sodium acetate / 1.0 M arginine, pH 5.2).

[0103] After affinity capture, several polishing steps were used to remove remaining impurities, including DNA, host cell proteins (HCPs), and leachable affinity ligands. Two chromatographic options were tested, including cation exchange and mixed-mode ion exchange. However, no process-specific host cell protein assays were available for characterization. Therefore, a commercially available ELISA was used as a surrogate, and LC-MS / MS quantification of specific HCPs was used as supplemental characterization. Additionally, 2D silver staining evaluation was used for quantitative characterization.

[0104] Figure 3 shows the evaluation of three potential downstream processes (DSPs). Three DSPs were identified based on the clearance of affinity ligand leachates. To assess product quality and clearance of process-related impurities, the same affinity elution pool was processed forward through each stream and compared for product recovery and clearance of CHO host cell proteins (HCPs; expressed in parts per million (ppm)). The eluate from each DSP process was further tested for product quality (Table below). [Table 5] Table Note: Product quality evaluation of three evaluated downstream processes. Eluates from the three DSPs (Figure 3) were evaluated for the effect of product quality. No effect on charge-based heterogeneity (IEX) or reversed-phase (RP) was observed. However, the sample from DSP-2, purified by a cation exchange polishing step, showed an increase in the % high molecular weight aggregates by size exclusion (SEC).

[0105] Based on the testing, a scale-up process was developed, which is shown in Figure 2. This process is simpler than that shown in Figure 1, but the yield was two-fold higher. The method described in Example 2, combined with a new cell culture system with a two- to three-fold increase in protein production, allowed for a four- to six-fold increase in overall antidote production, enabling a production scale of at least 10,000 L.

[0106] In Figure 2, the downstream process 200 uses a viral inactivation step (202) after collection (201) using only a detergent (Triton X-100) without solvent. The affinity capture step 203 uses a resin with an STI ligand, and the antidote is eluted with an arginine-containing elution buffer (pH 5). An optional ultrafiltration / diafiltration (UF / DF) step can be used after affinity capture, which can concentrate the protein and bring the pH to about 7.

[0107] In step 204, an anionic membrane is used to remove certain impurities (e.g., DNA, HCPs) from the sample. An example of an anionic membrane that has been tested is the Sartobind Q membrane, which did not exhibit any adverse effects on the antidote.

[0108] In step 205, an example of a type of mixed mode ion exchange is IEX mixed mode on CHT (ceramic hydroxyapatite). An example of an elution buffer is a phosphate buffer. This step is useful for removing STI leachate.

[0109] In step 206, hydrophobic separation can be achieved, for example, by octyl sepharose chromatography, and proteins can be eluted with a buffer containing NaCl, which is useful for removing remaining HCPs.

[0110] The table below shows the purification and recovery yields for several test runs. [Table 6]

[0111] Example 3: Characterization of the produced proteins In this example, methods were developed to characterize the protein products produced from the process developed above. The methods tested included isoelectric focusing (IEF), reduced reversed phase (Red-RP), and reduced peptide mapping (PMAP).

[0112] Isoelectric focusing was used to determine the charge heterogeneity of the antidote protein. Isoelectric focusing is an electrophoretic technique that separates proteins based on their isoelectric point (pI), or the pH at which they have no net charge. Isoelectric focusing was performed using gels ranging in pH from 3 to 10 and reagents for electrophoresis (Novex IEF gels) and staining with the Colloidal Blue Staining Kit (Invitrogen) according to the Life Technologies manufacturer's instructions. Results were compared to Serva 3-10 pI markers.

[0113] Test lots were diluted to 1.6 mg / mL in water and then made up 1:1 in IEF with sample buffers ranging from pH 3 to 10. 10 μL (8.0 μg) of each sample was loaded and electrophoresed at 100 V for 1 hour, followed by 200 V for 1 hour and 500 V for 30 minutes. Gels were fixed in fixative (Sigma-Aldrich) for 30 minutes, stained with Colloidal Blue stain for 1 hour, and destained overnight in water.

[0114] Reduced reversed-phase is an HPLC method that uses reversed-phase chromatographic separation for the detection of both full-length and truncated forms of proteins. Samples were buffer-exchanged into 6 M guanidine-HCl / 50 mM Tris pH 7.5, reduced with DTT, and then incubated at 50°C for 30 minutes. The samples were then alkylated with vinylpyridine in the dark at room temperature for 90 minutes, followed by quenching with 1 M DTT. This method utilizes an Agilent Zorbax C18 HPLC column to bind proteins to the stationary phase and a 15-95% gradient of a mobile phase of decreasing polarity (0.1% trifluoroacetic acid in acetonitrile) to separate proteins based on hydrophobicity. The UV wavelength is 214 nm.

[0115] The Lys-C digest peptide map was generated using UPLC-UV / MSE analysis of the Lys-C digest. This method provided greater than 98% sequence coverage and allowed characterization of post-translational modifications such as glycosylation, aspartic acid hydroxylation, and C-terminal lysine truncation. It also allowed monitoring of stability indicators such as asparagine deamidation and methionine oxidation. Each sample lot was reduced, alkylated, and treated with lysyl endopeptidase (Lys-C). Peptides generated by enzymatic digestion were separated by RP-UPLC and analyzed by mass spectrometry.

[0116] The table below shows the purified protein products and their percentages and types of protein isoforms detected from the sequences (Lot No. 1: using the subsequent process developed in Example 1; Lot No. 1: using the subsequent process developed in Example 2). Interestingly, compared to the process in Example 1, the process in Example 2 significantly reduced the percentage of SEQ ID NO: 9 (from 9.0% to 2.7%), likely due to increased purification efficiency. Light chain isoforms: [Table 7] Heavy chain isoforms: [Table 8-1] [Table 8-2] [Table 8-3]

[0117] Example 4: Validation of the scale-up process This example describes a process based on the process shown in Examples 1 and 2 for further validation.

[0118] Virus inactivation by surfactants A viral inactivation step was performed by adding 10% Triton X-100 to the collected protein. The collected protein corresponding to one Capto STI cycle (described below) was filtered from the collection tank through 4 x 30-inch Sartoguard NF filters used as prefilters into the 10% Triton X-100 addition tank, then through 3 x 20-inch Sartoguard NF filters 0.8 / 0.2 μm. After the 10% Triton X-100 addition, the pool was mixed and then transferred to the holding tank through 3 x 30-inch Sartoguard NF 0.8 / 0.2 μm filters. After the retention time was complete, the inactivated pool was loaded onto the Capto STI column through 6 x 30-inch Sartoguard NF filters 0.8 / 0.2 μm filters in series with 3 x 30-inch Sartopore 2 0.45 / 0.2 μm filters. This sequence was repeated until the four cycles performed during the Capto STI operation were completed. After the second cycle was filled, the 6x30 inch Sartoguard NF0.8 / 0.2 μm filter was replaced.

[0119] Capto STI Chromatography Affinity column chromatography was performed at ambient temperature using Capto STI resin. During the loading step, the product bound to the resin while contaminants were dispersed. Product recovery was achieved by disrupting protein interactions with a high concentration of arginine in the elution buffer. Prior to use, the column was rinsed with water for injection (WFI), washed with 100 mM phosphate (pH 3.0), and rinsed with WFI. The column was first pre-equilibrated with pre-equilibration buffer (20 mM Tris-HCl, pH 7.4), then equilibrated with equilibration buffer (20 mM Tris-HCl, 200 mM NaCl, pH 7.4). When the column pH and conductivity were within specifications, the column was loaded with Triton X-100-treated clarified harvest (HCCF). The HCCF material from each cycle was transferred directly from the viral inactivation holding tank to the column.

[0120] After loading, the column was washed with equilibration buffer and eluted with a high-arginine, low-pH elution buffer (elution buffer: 25 mM sodium acetate, 1.0 M arginine, pH 5.2). At the end of each cycle and before the next, the column was rinsed with WHI, washed again with 100 mM phosphate (pH 3.0), and rinsed again with WHI. All eluates from multiple cycles were pooled together. After collecting the final cycle, a buffer chase was performed to bypass the column to flush the eluted product from the subsequent line of the column into a collection container. After all cycles were completed, the column was rinsed with WFI, washed with 100 mM phosphate (pH 3.0), rinsed with WFI, pre-equilibrated, neutralized with equilibration buffer, and stored in an ethanol storage solution.

[0121] The Capto STI eluate pool was mixed with 1.0 M arginine HCl (pH 7.0) to a final concentration of 100 mM arginine HCl to stabilize the pool, and then concentrated and diafiltered onto a 10 KDa UltraCell Pelicon 3 membrane to remove most of the arginine and allow loading into the subsequent chromatography step.

[0122] Sartobind Q membrane chromatography Anion exchange membrane chromatography was performed using a Sartobind Q Jumbo cartridge at ambient temperature. During the loading phase, the product flowed through the membrane while contaminants bound. Prior to use, the cartridge was rinsed with equilibration buffer to remove the storage humectant. The cartridge was washed with 0.5 M NaOH. The cartridge was then equilibrated with equilibration buffer, and when the cartridge pH and conductivity were within specifications, it was loaded with concentrated, diafiltered Capto STI eluate. One cycle of loaded material was loaded directly from the post-UF / DF collection tank. After loading, the cartridge was washed with equilibration buffer. After collecting the product, a buffer chase was performed bypassing the cartridge to flush the product from the subsequent lines of the cartridge into the collection vessel.

[0123] Ceramic Hydroxyapatite Chromatography (CHT) Mixed-mode column chromatography was performed using CHT Type I 40 μm resin at ambient temperature. During the loading step, the product bound to the resin, while some contaminants were dispersed. The product was recovered by increasing the sodium concentration in a linear gradient. Prior to use, the column was washed with 1.0 M NaOH and rinsed with a small amount of equilibration buffer. The column was first pre-equilibrated with pre-equilibration buffer, then equilibrated with equilibration buffer, and loaded with Sartobind Q eluate when the column pH and conductivity were within specifications.

[0124] The load for each cycle was transferred directly from the collection tank to the column. After loading, the column was washed with equilibration buffer. A gradient was then run from 90% equilibration buffer (50 mM MES, 5 mM sodium phosphate, pH 7.0) to 90% elution buffer (50 mM MES, 5 mM sodium phosphate, 2 M sodium chloride, pH 7.0).

[0125] At the end of each cycle and before the next, the column was rinsed with equilibration buffer, then stripped with a high-phosphate post-elution wash buffer, rinsed with equilibration buffer, washed again with 1.0 M NaOH, and rinsed again with equilibration buffer. All eluates from several cycles were pooled together. After collecting the final cycle, a buffer chase was run to bypass the column to flush the eluted product down the line to a collection vessel. After all cycles were completed, the column was rinsed with equilibration buffer, washed with 1.0 M NaOH, and stored in the caustic / phosphate storage solution.

[0126] Octyl Sepharose 4FF chromatography Hydrophobic interaction chromatography was performed using ambient temperature Octyl Sepharose FF resin. During the loading phase, product flowed through the resin while contaminants bound. The column was washed with 1.0 M NaOH prior to use. The column was first pre-equilibrated with WFI and then equilibrated with equilibration buffer. When the column pH and conductivity were within specifications, the CHT eluate was loaded. The load from each cycle was transferred directly from the collection tank to the column. After loading, the column was washed with equilibration buffer. At the end of the cycle and before the next cycle, the column was stripped with WFI and washed again with 1.0 M NaOH. After collecting the final cycle, a buffer chase was performed to bypass the column to flush the product from the subsequent lines of the cartridge into the collection container. After all cycles were completed, the column was stripped with WFI, washed with 1.0 M NaOH, and stored in a caustic storage solution.

[0127] Planova 20N Viral Reduction Filtration (VRF) Viral reduction filtration was performed using a Planova 20N viral reduction filter. The filter was operated in dead-end mode, allowing the product to pass through the filter and any remaining viral particles to be retained by the fibers. Prior to use, the filter was flushed with Octyl Sepharose equilibration buffer. The product was then passed through the filter, followed by a further flush with equilibration buffer. The filter was tested for integrity and disposed of after use.

[0128] The VRF filtrate was concentrated on a 10 KDa Ultracel Pellicon 3 membrane. After partial concentration, it was diafiltered into the final formulation. The product was then concentrated to the final target concentration and collected.

[0129] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0130] The invention illustratively described herein can suitably be practiced in the absence of any element(s), limitation(ies), not specifically disclosed herein. Thus, for example, terms such as "comprise," "include," and "contain" shall be read expansively and without limitation. Furthermore, the terms and expressions used herein are used as terms of description rather than limitation, and there is no intention to use such terms and expressions to exclude any equivalents of the shown and described features or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed.

[0131] Thus, while the present invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that modifications, improvements, and variations of the invention embodied therein and disclosed herein may be resorted to by those skilled in the art, and that such improvements and variations are within the scope of the present invention. The materials, methods, and examples provided herein are representative of preferred embodiments and are illustrative and do not limit the scope of the invention.

[0132] The invention is described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a condition or negative limitation removing any subject matter from the genus, regardless of whether the removed material is specifically recited herein.

[0133] Additionally, although features or aspects of the invention have been described in terms of Markush groups, one of skill in the art will recognize that the invention may also be described in terms of any individual member or subgroup of members of a Markush group.

[0134] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety, as if each were individually incorporated by reference to the same extent as if each were individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

[0135] While the present disclosure has been described in conjunction with the above embodiments, it should be understood that the foregoing description and examples are intended to be illustrative, and not limiting, of the scope of the present disclosure. Other aspects, advantages, and modifications within the scope of the present disclosure will be apparent to those skilled in the art to which this disclosure pertains.

Claims

1. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polypeptide portion of a double-chain polypeptide processed from a polypeptide expressed from a polynucleotide comprising the nucleic acid sequence of SEQ ID NO: 7, wherein each of the double-chain polypeptides comprises a light chain and a heavy chain, and 0.5 to 10% by weight of the heavy chain consists of the amino acid sequence of SEQ ID NO:

9.

2. The pharmaceutical composition described in claim 1, wherein at least 35% by weight of the light chain comprises the amino acid sequence of SEQ ID NO:

4.

3. A pharmaceutical composition described in claim 1 or 2, wherein 20 to 50% by weight of the heavy chain consists of the amino acid sequence of SEQ ID NO:

5.

4. A pharmaceutical composition described in any one of claims 1 to 3, wherein 40 to 60% by weight of the heavy chain consists of the amino acid sequence of SEQ ID NO:

8.

5. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and polypeptide portions of a double-chain polypeptide, each polypeptide portion comprising a light chain and a heavy chain, at least 35% by weight of the light chains comprise the amino acid sequence of SEQ ID NO:4; 20 to 50% by weight of the heavy chain consists of the amino acid sequence of SEQ ID NO:5; 40 to 60% by weight of the heavy chain consists of the amino acid sequence of SEQ ID NO: 8; A pharmaceutical composition, wherein 0.5 to 10% by weight of the heavy chain consists of the amino acid sequence of SEQ ID NO:

9.

6. A pharmaceutical composition described in any one of claims 1 to 5, wherein 0.5 to 5% by weight of the heavy chain consists of the amino acid sequence of SEQ ID NO:

9.

7. The pharmaceutical composition described in claim 6, wherein 0.5 to 3% by weight of the heavy chain consists of the amino acid sequence of SEQ ID NO:

9.

8. A pharmaceutical composition described in any one of claims 1 to 5, wherein 2 to 10% by weight of the heavy chain consists of the amino acid sequence of SEQ ID NO:

9.

9. A pharmaceutical composition described in any one of claims 1 to 8, wherein 0.1 to 1.5% by weight of the heavy chain consists of the amino acid sequence of SEQ ID NO:

10.

10. A pharmaceutical composition described in any one of claims 1 to 9, wherein 2 to 8% by weight of the heavy chain consists of the amino acid sequence of SEQ ID NO:

11.

11. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is freeze-dried.

12. The pharmaceutical composition of claim 1, further comprising L-arginine HCl or L-arginine acetate.

13. A pharmaceutical composition described in any one of claims 1 to 12, further comprising sucrose.

14. A pharmaceutical composition described in any one of claims 1 to 13, further comprising mannitol.

15. A pharmaceutical composition according to any one of claims 1 to 14 for use in reversing or inhibiting anticoagulation in patients undergoing anticoagulant therapy with a factor Xa inhibitor.

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