Preparation of Factor XA Derivatives

Abstract

The present disclosure provides a method for large-scale manufacturing of fXa-derived proteins to obtain a high yield of a high-purity protein product. The method may include adding a detergent to a sample containing a polynucleotide construct encoding the protein, and purifying the protein by affinity chromatography, ion exchange and mixed mode chromatography and hydrophobic interaction based on soybean trypsin inhibitor (STI).

Description

[0001] This application is a divisional application of an invention patent application which entered the Chinese national phase on January 3, 2019, with application number 201780041530.2, application date June 19, 2017, and invention name “Preparation of Factor XA Derivatives”.

[0002] Cross-references to related patent applications

[0003] 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 Art

[0004] Modified derivatives of the factor Xa (fXa) protein have been developed that can be used as antidotes for anticoagulants that target fXa. The derivatives are being developed as universal reversal agents for patients who are anticoagulated with oral or injectable factor Xa inhibitors and need to have the anticoagulation reversed. Summary of the invention

[0005] The present disclosure provides a method for large-scale manufacture of fXa detoxification polypeptides to obtain a high yield of a high-purity protein product. In one embodiment, a method for preparing a polypeptide product expressed by a polynucleotide construct is provided, the polynucleotide construct including SEQ ID NO:7 nucleic acid sequence or encoding and by SEQ ID NO:7 encoded amino acid sequence having at least 90% sequence identity of the amino acid sequence. The method may include adding a detergent to a sample containing a polynucleotide construct, and purifying the encoded detoxification protein by affinity chromatography, ion exchange and mixed mode chromatography and / or hydrophobic interaction based on soybean trypsin inhibitor (STI).

[0006] In one embodiment, a method for preparing a polypeptide product expressed by a polynucleotide construct is provided, wherein the polynucleotide construct includes a nucleic acid sequence of SEQ ID NO:7 or a nucleic acid sequence encoding an amino acid sequence having at least 90% sequence identity with the amino acid sequence encoded by SEQ ID NO:7, the method comprising adding a detergent to a sample containing the polynucleotide construct and the polypeptide product expressed by the polynucleotide construct; loading the sample into a soybean trypsin inhibitor (STI)-based affinity chromatography, and eluting the polypeptide with a first elution buffer to produce a first elution sample, wherein the loaded sample does not contain an organic solvent; loading the first elution sample into ion exchange and mixed mode chromatography, and eluting the polypeptide with a second elution buffer comprising at least 1 M inorganic salt to produce a second elution sample; and loading the second elution sample into a hydrophobic interaction chromatography, 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.

[0007] In some embodiments, the detergent comprises Triton X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether). In some embodiments, the first elution buffer comprises 0.5M to 2M arginine. In some embodiments, the first elution buffer has a pH of about 5 to 5.4. In some embodiments, the ion exchange and mixed mode chromatography comprises ceramic hydroxyapatite type I chromatography.

[0008] In some embodiments, the second elution buffer comprises an inorganic salt of at least 2 M. In some embodiments, the inorganic salt is sodium chloride. In some embodiments, the hydrophobic interaction chromatography comprises octyl sepharose chromatography.

[0009] In some embodiments, the method further comprises a purification step using anion exchange chromatography. In some embodiments, the anion exchange chromatography comprises Sartobind TM Ion exchange membrane.

[0010] In some embodiments, the method further comprises filtering one or more samples with a nanofishnet membrane filter. In some embodiments, filtering with a nanofishnet membrane filter is performed before loading the sample into the STI-based affinity chromatography.

[0011] In some embodiments, the purified sample contains less than about 1% of contaminating proteins that are not expressed by the polynucleotide construct. In some embodiments, the polypeptide product is expressed in a cell containing a polynucleotide construct. In some embodiments, the cell is grown in a culture medium under conditions that produce at least 100 mg of polypeptide product per liter of culture medium. In some embodiments, the cell is grown in a culture medium under conditions that produce at least 200 mg of polypeptide product per liter of culture medium. In some embodiments, the purified sample contains greater than about 50% of the polypeptide product produced in the culture medium. In some embodiments, the purified sample contains greater than about 100 mg of polypeptide product from each liter of culture medium product.

[0012] 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 the purified sample have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 5. In some embodiments, about 5%-95% of the heavy chains consisting of the amino acid sequence of SEQ ID NO: 5 have two O-linked glycosylations, and about 5%-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%-80% of the polypeptide products in the 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%-12% of the polypeptide products in the purified sample have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 9. In some embodiments, about 0.1%-1.5% of the polypeptide products in the purified sample have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 10. In some embodiments, about 2%-8% of the polypeptide products in the purified sample have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 11. In some embodiments, about 35%-60% of the polypeptide products in the purified sample have a light chain consisting of the amino acid sequence of SEQ ID NO:4.

[0013] In one embodiment, a polypeptide prepared by the method of any one of the embodiments is also provided.

[0014] In one embodiment, a pharmaceutical composition is provided, comprising a pharmaceutically acceptable carrier and a polypeptide portion of a double-chain polypeptide, wherein: about 35%-60% of the double-chain polypeptide has a light chain consisting of the amino acid sequence of SEQ ID NO:4; about 20%-60% of the double-chain polypeptide has a heavy chain consisting of the amino acid sequence of SEQ ID NO:5; about 40%-60% of the double-chain polypeptide has a heavy chain consisting of the amino acid sequence of SEQ ID NO:8; and less than 10% of the double-chain polypeptide has a heavy chain consisting of the amino acid sequence of SEQ ID NO:9.

[0015] In some embodiments, less than 5% of the double-chain polypeptides have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 9. In some embodiments, less than 3% of the double-chain polypeptides have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 9. In some embodiments, about 0.1%-1.5% of the double-chain polypeptides have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 10. In some embodiments, about 2%-8% of the double-chain polypeptides have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 11. In some embodiments, about 30%-70% of the heavy chains consisting of the amino acid sequence of SEQ ID NO: 5 have two O-linked glycosylations. In some embodiments, about 30%-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.

[0016] In some embodiments, the preparation is lyophilized. In some embodiments, the composition further comprises L-arginine hydrochloride or L-arginine acetate. In some embodiments, the composition further comprises sucrose. In some embodiments, the composition further comprises mannitol.

[0017] In one embodiment, a method for reversing or inhibiting anticoagulation in a patient undergoing anticoagulation therapy with a factor Xa inhibitor is also provided, comprising administering to the patient the pharmaceutical composition of any one of the embodiments of the present disclosure. DETAILED DESCRIPTION

[0018] definition

[0019] The following description sets forth exemplary embodiments of the present technology. However, it should be appreciated that such description is not intended to limit the scope of the present disclosure, but is provided as a description of exemplary embodiments.

[0020] All numerical indications, such as pH, temperature, time, concentration, and molecular weight (including ranges) are approximate values ​​with variations of (+) or (-) 0.1% or 10% increments. It should be understood that, although not always explicitly stated, all numerical indications are preceded by the term "about". It should also be understood that, although not always explicitly stated, the reagents described herein are merely exemplary and equivalents of these reagents are known in the art.

[0021] As used in this 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.

[0022] As used herein, the term "comprising" is intended to mean that compositions and methods include the described elements, but do not exclude other elements. "Substantially consisting of" when used to define compositions and methods should mean excluding other elements that are of any importance to the combination for the intended use. Therefore, a composition consisting essentially of the elements defined herein will not exclude trace impurities and pharmaceutically acceptable carriers from separation and purification methods, such as phosphate buffered saline, preservatives, etc. "Consisting of" should mean excluding elements of other ingredients exceeding trace amounts and basic method steps for applying the compositions of the present disclosure. Embodiments defined by each of these transitional terms fall within the scope of the present disclosure.

[0023] The terms "protein" and "polypeptide" are used interchangeably and, in their broadest sense, refer to compounds of two or more subunit amino acids, amino acid analogs or peptide mimetics. The subunits may be linked by peptide bonds. In another embodiment, 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 can constitute the sequence of a protein or the sequence of a peptide. As used herein, the term "amino acid" refers to natural amino acids and / or non-natural or synthetic amino acids, including glycine and both D-type optical isomers and L-type optical isomers, amino acid analogs and peptidomimetics. The single-letter abbreviations and three-letter abbreviations of naturally occurring amino acids are listed below.

[0024] "Factor Xa" or "fXa" or "fXa protein" is a serine protease in the blood coagulation pathway, which is produced by inactive factor X (fX, SEQ ID NO.1, Table 1). The nucleotide sequence encoding human factor X ("fX") can be found in the gene bank by accession number "NM_000504". When the first 52 residues of the catalytic cleavage heavy chain are activated to fXa. FXa contains a light chain and a heavy chain. The first 45 amino acid residues of the light chain (residues 1 to 45 of SEQ ID NO.1) are called Gla domains because they contain 11 post-translationally modified γ-carboxyglutamic acid residues (Gla). It also contains a short (6 amino acid residues) aromatic stacking sequence (residues 40-45 of SEQ ID NO.1). Chymotrypsin digestion selectively removes residues 1-44, thereby producing fXa without Gla domain. The serine protease catalytic domain of fXa is located at the C-terminal heavy chain. The heavy chain of fXa is highly homologous to other serine proteases such as thrombin, trypsin, and activated protein C.

[0025] "Native fXa" or "wild-type fXa" refers to fXa naturally present in plasma or isolated in its original unmodified form, which processes the biological activity of activated prothrombin, thereby promoting the formation of blood clots. The term includes naturally occurring polypeptides isolated from tissue samples and recombinantly produced fXa. "Active fXa" refers to fXa with procoagulant activity of activated prothrombin. "Active fXa" can be natural fXa or modified fXa that retains procoagulant activity.

[0026] As used herein, "fXa antidote", "antidote" or "fXa derivative" refers to a modified fXa protein that does not compete with fXa in assembling into a prothrombinase complex, and has reduced procoagulant activity or no procoagulant activity, and also binds and / or substantially neutralizes anticoagulants, such as fXa inhibitors. In some aspects, the "procoagulant activity" of fXa protein or fXa derivative refers to the enzymatic activity carried by wild-type active fXa polypeptides. Examples of fXa derivatives are provided in U.S. Patent No. 8,153,590 and PCT Publications WO2009 / 042962 and WO2010 / 056765, and are further provided herein, such as SEQ ID NO: 2 and 3 and their bioequivalents.

[0027] "Enzymatic activity" of a fXa polypeptide or derivative thereof refers to the ability of the polypeptide to catalyze a biochemical reaction with a substrate by directly interacting with the substrate.

[0028] SEQ ID NO:2 contains 3 mutations relative to wild-type fXa. The first mutation is the deletion of 6-39aa in the Gla domain of fX. The second mutation is to replace the activation peptide sequence 143-194aa with -RKR-. This produces -RKRRKR-(SEQ ID NO:6) joints connecting light chain (SEQ ID NO:4) and heavy chain (SEQ ID NO:5). During secretion, this joint is cleaved to produce a double-chain polypeptide SEQ ID NO:3 (r- antidote). The third mutation is that the active site residue S379 mutates to an Ala residue. This amino acid substitution corresponds to amino acids 296 and 290 of SEQ ID NO:1 and 3, respectively.

[0029] An example of an antidote is "r-antidote", which refers to a double-chain polypeptide processed product of SEQ ID NO:2 after the cleavage linker. This is represented by SEQ ID NO:3. R-antidote is disclosed in, for example, US 8,153,590, the contents of which are incorporated herein by reference. R-antidote includes a light chain (SEQ ID NO.4) and a heavy chain (SEQ ID NO.5), which is connected to a single disulfide bond between cysteine ​​98 (Cys98) of the light chain and cysteine ​​108 (Cys108) of the heavy chain. Like wild-type fXa, in some production batches, the r-antidote undergoes post-translational modification, thereby causing glycosylation at certain amino acid residues, such as Ser56, Ser72, Ser76 and Thr82 of the light chain and Thr249 of the heavy chain, and the modified residue of the light chain, i.e., (3R)-3-hydroxy Asp at Asp29. Furthermore, in addition to the interchain disulfide bonds, intrachain disulfide bonds can also be formed between cysteines 16 and 27, 21 and 36, 38 and 47, 55 and 66, 62 and 75, and 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.

[0030] Table 1. Inactive human factor X polypeptide sequence (SEQ ID NO: 1)

[0031]

[0032] Table 2. Peptide sequence of r-Antidote precursor before removal of -RKRRKR- (SEQ ID NO.6) linker (SEQ ID NO:2)

[0033]

[0034] Table 3 Polypeptide sequence of human factor Xa triple mutant after removal of -RKRRKR- (SEQ ID NO.6) linker (SEQ ID NO: 3)

[0035]

[0036]

[0037] Table 4. Nucleotide sequence encoding r-Antidote precursor (SEQ ID NO: 7)

[0038]

[0039] Other example antidotes can be r-antidotes (or its precursor, represented by SEQ ID NO: 2) bioequivalents, or alternatively polypeptides with certain sequence identities to SEQ ID NO: 3. On the one hand, such bioequivalents retain the structural features of SEQ ID NO: 3, i.e., modified active sites and missing or modified Gla domains. On the other hand, such bioequivalents retain the functional features of SEQ ID NO: 3, i.e., do not compete with fXa in terms of assembling into prothrombinase complexes and have reduced procoagulant activity (e.g., enzymatic activity or catalytic activity) or no procoagulant activity (e.g., enzymatic activity or catalytic activity).

[0040] The term "active site" refers to the part of an enzyme or antibody where a chemical reaction occurs. A "modified active site" is an active site that has been structurally modified to provide increased or decreased chemical reactivity or specificity to the active site. Examples of active sites include, but are not limited to, a catalytic domain comprising amino acid residues 235-488 of human factor X and a catalytic domain comprising amino acid residues 195-448 of human factor Xa. Examples of modified active sites include, but are not limited to, a catalytic domain comprising amino acid residues 195-448 in SEQ ID NO: 1 of human factor Xa, wherein at positions Arg306, Glu310, Arg347, Lys351, Lys414, or Arg424 have at least one amino acid substitution.

[0041] Preparation of antidote

[0042] Experimental example (embodiment 1 and 2) demonstrates the development of culture and purification method for preparing fXa antidote.Cultivated cells include polynucleotide constructs, which include SEQ ID NO:7 nucleic acid sequence or encoding and by SEQ ID NO:7 encoded amino acid sequence has at least 90% sequence identity (or at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity) of the nucleic acid sequence of the amino acid sequence.Cell is typically Chinese hamster ovary (CHO) cell.

[0043] A purification method inFigure 1 . After the cells are harvested (step 101) and clarified using a depth filter to remove high molecular weight (HWM) impurities, the sample is concentrated (e.g., about 10 times) (step 102). The concentration step can use regenerated cellulose, but is not limited to this. In step 103, virus inactivation (e.g., with a detergent / solvent; such as 1% Triton X-100, 0.3% tributyl phosphate (final concentration)) can be performed to inactivate enveloped viruses in the cell culture. After removing the virus, 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 antidotes. In step 107, hydrophobic interaction resins can be used to further remove remaining host cell proteins. Optionally, after these purification steps, a final virus removal filtration step can be used to remove any remaining viruses.

[0044] In another embodiment, the purification process is as Figure 2 In some embodiments, the method comprises adding a detergent to a sample containing a polynucleotide construct (e.g., SEQ ID NO: 7) and a polypeptide product expressed by the polynucleotide construct (e.g., SEQ ID NO: 3). In some embodiments, the detergent comprises Triton X-100. In some embodiments, no solvent is used to treat the sample before the sample undergoes subsequent affinity purification. In some embodiments, no organic solvent is used to treat the sample before the sample undergoes subsequent affinity purification. In some embodiments, tributyl phosphate is not added to the sample before the sample undergoes subsequent affinity purification.

[0045] In some embodiments, the sample is then loaded into a soybean trypsin inhibitor (STI)-based affinity chromatography and eluted with an elution buffer to produce an eluted sample. In some embodiments, the loaded sample is not treated with an organic solvent or does not contain an organic solvent.

[0046] "STI" or "soybean trypsin inhibitor" refers to a trypsin inhibitor isolated from soybean or its bioequivalent. Trypsin inhibitor is about 20 kDa in size and reduces trypsin (a proteolytic enzyme) as well as 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), whose GenBank accession number is NP_001238611.

[0047] STI can be fixed on solid support resin for purification of certain proteins. In addition to soybean trypsin inhibitor, other trypsin inhibitor proteins, such as trypsin inhibitor proteins isolated from serum, lima beans, bovine pancreas or ovomucoid, or modified forms thereof, can also be used. Certain protease inhibitors, particularly serine protease inhibitors, are also contemplated, which can be used to prepare affinity resins for the purpose of purifying antidotes. The antidotes can then be eluted with a buffer containing arginine. In some embodiments, the elution buffer comprises 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 0.5M to 2M arginine, or about 0.7M to 1.5M arginine, or 0.8M to 1.2M arginine. In some embodiments, the pH of the elution buffer is 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, pH 5.2.

[0048] In some embodiments, the sample is then loaded into ion exchange and mixed mode chromatography. Non-limiting examples of ion exchange and mixed mode chromatography include ceramic hydroxyapatite type I chromatography. The sample can then be eluted with an elution buffer comprising an inorganic salt. In some embodiments, the inorganic salt is sodium chloride or potassium chloride. In some embodiments, the concentration of salt in the elution buffer is at least 0.5M, 1M, 1.5M, 2M, 2.5M or 3M.

[0049] In some embodiments, elution needs to use the gradient generated between the equilibrium buffer and the elution buffer. In some embodiments, the equilibrium buffer includes a lower concentration of salt, for example, less than 0.2M, less than 0.1M, less than 50mM, less than 20mM, less than 10mM. In some embodiments, the gradient includes a salt concentration increase of at least 5 times, or 10 times, or 20 times, or 50 times, or 100 times. In some embodiments, the equilibrium buffer includes 50mM MES (2-(N-morpholino) ethanesulfonic acid), 5mM sodium phosphate, pH 7.0. In some embodiments, the elution buffer includes 50mM MES, 5mM sodium phosphate, 2M sodium chloride, pH 7.0. In some embodiments, the gradient starts with about 90% of the equilibrium buffer and ends with about 90% of the elution buffer.

[0050] In some embodiments, the sample is further loaded into a hydrophobic interaction chromatography. In some embodiments, the hydrophobic interaction chromatography comprises octyl sepharose chromatography. In some embodiments, the sample is eluted with an elution buffer comprising at least 2 mM sodium chloride, or alternatively at least 1 M, 1.5 M, 2.5 M, 3 M, 4 M or 5 M sodium chloride.

[0051] In some embodiments, one of the intermediate samples is further subjected to a purification step using anion exchange chromatography. In some embodiments, the anion exchange chromatography comprises Sartobind TM Ion Exchange Membranes. In some embodiments, anion exchange chromatography is applied after affinity chromatography.

[0052] In some embodiments, one of the intermediate samples is further filtered using a nanofishnet membrane filter. In some embodiments, after applying the nanofishnet membrane filtration, the sample is loaded into STI-based affinity chromatography.

[0053] In some embodiments, the purified sample contains less than about 1% of contaminating proteins not expressed by the polynucleotide construct, such as host cell proteins or STI released from an affinity resin / column.

[0054] The purification process of any one of the above embodiments can recycle at least about 50% (or at least about 40%, 45%, 55%, 60%, 65%, 70%, 75%, 80% or 85%) of the detoxification protein expressed in the cell culture. In some embodiments, the cells are grown in the culture medium under the conditions of producing at least 100 mg of polypeptide product per liter of culture medium. In some embodiments, the cells are grown in the culture medium under the conditions of producing 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 medium. In some embodiments, the purified sample contains greater 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 per liter of culture medium product.

[0055] Antidote isomers in purified products

[0056] According to the method described in any one of the embodiments of the present disclosure, the purified antidote product is considered to include light chains and heavy chains. However, the culture and purification process may introduce changes in the actual protein.

[0057] As shown in Example 3, about 20% to 50% of the polypeptide products in the purified sample have intact heavy chains (SEQ ID NO: 5). Of these polypeptide products with intact heavy chains, about 5%-95% of the heavy chains have two O-linked glycosylations, and about 5%-95% of the heavy chains have one O-linked glycosylation.

[0058] In some embodiments, at least about 20%, 25%, 30%, 35%, 40% or 45% of the polypeptide products in the purified sample have complete heavy chains. In some embodiments, no more than about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% of the polypeptide products have complete heavy chains.

[0059] In some embodiments, about 40%-80% of the polypeptide products in the purified sample have a heavy chain with a C-terminal lysine deleted (SEQ ID NO:8). In some embodiments, SEQ ID NO:8 accounts for at least about 20%, 25%, 30%, 35%, 40%, 45%, or 50% in the purified sample. In some embodiments, SEQ ID NO:8 accounts for no more than about 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% in the purified sample. In some embodiments, at least about 90% of the heavy chain of SEQ ID NO:8 has an O-linked glycosylation.

[0060] In some embodiments, about 2%-12% of the polypeptide products in the purified sample have a heavy chain with 13 C-terminal amino acid residues deleted (SEQ ID NO:9). In some embodiments, SEQ ID NO:9 accounts for at least about 0.5%, 1%, 1.5%, 2%, 2.5%, 3% or 5% in the purified sample. In some embodiments, SEQ ID NO:9 accounts for no more than about 2%, 3%, 4%, 5%, 7%, 10%, 15%, 17% or 20% in the purified sample.

[0061] In some embodiments, about 0.1%-1.5% of the polypeptide products in the purified sample have a heavy chain with 14 C-terminal amino acid residues deleted (SEQ ID NO: 10). In some embodiments, SEQ ID NO: 10 accounts for at least about 0.05%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3% or 0.5% in the purified sample. In some embodiments, SEQ ID NO: 10 accounts for no more than about 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5% or 3% in the purified sample.

[0062] In some embodiments, about 2%-8% of the polypeptide products in the purified sample have a heavy chain with 15 C-terminal amino acid residues deleted (SEQ ID NO: 11). In some embodiments, SEQ ID NO: 11 accounts for at least about 0.5%, 1%, 1.5%, 2%, 2.5%, 3% or 5% in the purified sample. In some embodiments, SEQ ID NO: 11 accounts for no more than about 5%, 6%, 7%, 8%, 9% or 10% in the purified sample.

[0063] Embodiment 3 also shows that about 35%-60% of the polypeptide products in the purified sample have complete light chains (SEQ ID NO:4), while some others can be modified or truncated. In some embodiments, the amount of the complete light chain in the total light chain is at least about 20%, 25%, 30%, 35%, 40%, 45% or 50%. In some embodiments, the amount of the complete light chain in the total light chain is no more than about 45%, 50%, 55%, 60%, 65%, 70%, 80% or 90%.

[0064] In some embodiments, the present disclosure provides a pharmaceutical preparation comprising a pharmaceutically acceptable carrier and a polypeptide portion of a double-chain polypeptide, wherein about 35%-60% of the double-chain polypeptide has a light chain consisting of the amino acid sequence of SEQ ID NO: 4. In some embodiments, about 20%-50% of the double-chain polypeptide has a heavy chain consisting of the amino acid sequence of SEQ ID NO: 5. In some embodiments, about 40%-80% of the double-chain polypeptide has a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8. In some embodiments, less than 10%, 9%, 8%, 7%, 6%, 5%, 4% or 3% of the double-chain polypeptide has a heavy chain consisting of the amino acid sequence of SEQ ID NO: 9. In some embodiments, about 0.1%-1.5% of the double-chain polypeptide has a heavy chain consisting of the amino acid sequence of SEQ ID NO: 10. In some embodiments, about 2%-8% of the double-chain polypeptide has a heavy chain consisting of the amino acid sequence of SEQ ID NO: 11.

[0065] In some embodiments, about 5%-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%-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.

[0066] Preparation, administration and dosage

[0067] Also provided are preparations prepared with purified detoxification protein products. In some embodiments, aqueous preparations suitable for freeze drying are provided. In one embodiment, the preparation includes an antidote and a solubilizing agent, a stabilizer and a crystallizing agent. The preparation may also include a surfactant and / or a buffer. In some aspects, the presence of each of these agents prevents the antidote from collapsing during freeze drying, for example, at a freeze drying temperature higher than -40°C, -30°C, -20°C, -10°C, 0°C, 5°C, 10°C or 15°C, up to 20°C or 25°C.

[0068] "Crystallization component" refers to molecules that form a crystallization matrix in a formulation including a polypeptide during the freeze-drying process. Non-limiting examples of crystallization components include mannitol and glycine.

[0069] In some aspects, the crystalline component is mannitol (e.g., crystalline mannitol). On the one hand, the concentration of the crystalline component in the aqueous formulation is at least 1% (w / v). On the one hand, 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). On the one hand, the concentration of the crystalline component in the aqueous formulation is not higher than 8%, or optionally not higher than 7%, 6.5%, 6%, 5.5%, 5%, 4.5% or 4% (w / v). On the one hand, 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 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).

[0070] In some respects, solubilizing agents are included in aqueous formulations. 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., active ingredient) in solution. Non-limiting examples of solubilizing agents include arginine and citrate. On the one hand, the solubilizing agent is arginine. On the one hand, the solubilizing agent is citrate.

[0071] The presence of a solubilizing agent can be used to keep fXa polypeptides soluble and stable in a formulation. In some aspects, the concentration of a solubilizing agent (such as arginine) is at least 10mM, or alternatively at least 20mM, 25mM, 30mM, 36mM or 40mM. In some aspects, the concentration of a solubilizing agent (such as arginine) is not higher than 100mM, 96mM, 90mM, 80mM, 70mM, 60mM or 50mM. In some aspects, the concentration of a solubilizing agent is about 10mM or 20mM to about 60mM, about 10mM or 20mM to about 55mM, about 35mM to about 55mM, about 40mM to about 50mM, about 41mM to about 49mM, about 42mM to about 48mM, about 43mM to about 47mM, about 44mM to about 46mM, or about 40mM, 45mM or 50mM. It should be noted that as used herein, the term arginine refers to the amino acid as well as its salts (e.g., arginine hydrochloride). Arginine has a molecular weight of about 174.2 Daltons and arginine hydrochloride (e.g., L-arginine hydrochloride, L-arginine acetate) has a molecular weight of about 210.7 Daltons.

[0072] In one embodiment, the solubilizing agent is citrate or a salt thereof. The salt of citrate is sodium citrate. On the one hand, citrate comprises a concentration of about 1.0 mM to about 200.0 mM. On the other hand, the concentration of citrate is about 25 mM. On the other hand, the concentration of citrate is about 50 mM. In another embodiment, the concentration of citrate is about 5 mM, 10 mM, or 20 mM. In another embodiment, citrate comprises a concentration of about 0.05 M to about 0.2 M.

[0073] In some aspects, stabilizers are included in aqueous formulations. The term "stabilizer" represents a pharmaceutically acceptable excipient that protects active ingredients (such as fXa derived polypeptides) and / or formulations from chemical degradation and / or physical degradation during manufacture, storage, and application. Examples of stabilizers may include sucrose, arginine, citrate, mannitol, trehalose, glycine, sodium chloride, dextran, and glucose. On the one hand, stabilizer is sucrose.

[0074] In one aspect, the concentration of the stabilizer (e.g., sucrose) in the aqueous formulation is at least about 0.5% (w / v). In one aspect, 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 about 2% (w / v). In one aspect, the concentration of the stabilizer (e.g., sucrose) in the aqueous formulation is no more than 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 from about 1% to about 5%, or from about 1% to about 4%, or from about 1% to about 3%, or from about 1.5% to about 2.5%, or from about 1.6% to about 2.4%, or from about 1.7% to 2.3%, or from about 1.7% to about 2.2%, or from about 1.9% to about 2.1%, or about 1%, 1.5%, 2%, 2.5% or 3% (w / v).

[0075] In some aspects, the aqueous formulation may further comprise a surfactant, a buffer, a tonicity agent, a cryoprotectant, a surfactant, a lyoprotectant, a preservative, or a combination thereof.

[0076] In some aspects, the aqueous formulation has a pH of 6 or more, or 6.5 or more, or 7 or more, or 7.5 or more. In some aspects, the pH is no greater than 9, 8.5, or 8. In some aspects, the pH is between 6 and 9, between 6.5 and 8.5, between 7 and 8.5, between 7.5 and 8.2, between 7.6 and 8.1, between 7.7 and 7.9, or about 7.5, 7.6, 7.7, 7.8, 7.9, or 8.

[0077] In one aspect, the aqueous formulation comprises about 45 mM arginine, about 2% sucrose (w / v), about 5% mannitol (w / v), and about 10 mg / mL of double-chain r-Antidote, wherein the formulation has a pH of about 7.8. In one aspect, the aqueous formulation comprises about 45 mM arginine, about 2% sucrose (w / v), about 5% mannitol (w / v), and about 20 mg / mL of double-chain r-Antidote, wherein the formulation has a pH of about 7.8. In one aspect, the aqueous formulation comprises about 45 mM arginine, about 2% sucrose (w / v), about 5% mannitol (w / v), and about 40 mg / mL of double-chain r-Antidote, wherein the formulation has a pH of about 7.8. In one aspect, the aqueous formulation further comprises 0.01%-0.02% (w / v) of polysorbate 80 and a buffer.

[0078] In some aspects, a lyophilized composition prepared by lyophilizing the aqueous formulation of the present disclosure is also provided.Based on the concentration of each pharmaceutical agent in the aqueous formulation, the relative content of the pharmaceutical agent in the lyophilized composition can be easily determined.

[0079] On the one hand, the lyophilized composition comprises at least 5%, or alternatively at least 10%, 15%, 20%, 25%, 30% or 35% (w / w) of fXa antidote. Then, in other main ingredients, for example, the weight ratio of L-arginine hydrochloride: sucrose: mannitol can be in the range of (0.5-1.4): (1-3): (2-6). In some aspects, the weight ratio of L-arginine hydrochloride: 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 aspects, the lyophilized composition further comprises a solid portion of a surfactant and / or a buffer.

[0080] The present disclosure also relates to a method of treating, preventing or reducing bleeding in a subject receiving anticoagulant therapy with a fXa inhibitor, the method comprising administering to the subject an effective amount of the lyophilized formulation when the lyophilized formulation is dissolved in a suitable solvent. It is contemplated that the antidote or derivative of the present disclosure may be a short-duration drug to be used in selective or emergency situations that can safely and specifically neutralize the conventional anticoagulant properties of fXa inhibitors without causing harmful hemodynamic side effects or exacerbation of the proliferative vascular response to injury.

[0081] As used herein, the terms "treating", "treatment" and the like are used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect may be preventive, in terms of completely or partially preventing a condition or its signs or symptoms, and / or may be therapeutic, in terms of partially or completely curing a condition and / or adverse effects attributable to the condition.

[0082] "Treatment" also encompasses any treatment of a mammalian condition, and includes: (a) preventing the condition from occurring in a subject who may be susceptible to the condition but who may not have yet been diagnosed as having the condition, e.g., preventing bleeding in a patient who has had an overdose of an anticoagulant; (b) inhibiting the condition, i.e., stopping its development, e.g., inhibiting bleeding; or (c) alleviating or relieving the condition, e.g., reducing bleeding.

[0083] As used herein, "treatment" also includes systemic amelioration of symptoms associated with a pathology and / or delay in the onset of symptoms. The clinical and subclinical evidence of "treatment" will vary with the pathology, the individual, and the treatment.

[0084] "Administration" can be performed continuously or intermittently throughout the treatment process in a single administration. Methods for determining the most effective mode of administration and dosage are known to those skilled in the art and will vary with the composition used for therapy, the purpose of therapy, the target cells treated, and the subject treated. Single or multiple administrations can be performed using the administration level and mode selected by the treating physician. Suitable dosage formulations and methods for administering medicaments are known in the art. The "subject" of diagnosis or treatment is a cell or mammal, including humans. Non-human animal subjects of diagnosis or treatment include, for example, rodents (such as rats, mice), canines (such as dogs), lagomorphs (such as rabbits), livestock, competitive animals, and pets.

[0085] The agents and compositions of the present disclosure may be used in the manufacture of medicaments and may be used to treat humans and other animals by administration according to conventional procedures, such as active ingredients in pharmaceutical compositions.

[0086] The medicaments of the present disclosure can be administered for therapy by any suitable route, in particular by parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. It will also be understood that the preferred route will vary with the condition and age of the recipient and the disease being treated.

[0087] The phrase "pharmaceutically acceptable polymer" refers to a group of compounds that can be conjugated to one or more polypeptides described herein. It is contemplated that conjugation 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, polyvinyl pyrrolidone, polyvinyl alcohol, cellulose derivatives, polyacrylates, polymethacrylates, sugars, polyols, and mixtures thereof.

[0088] An "anticoagulant" or "anticoagulant" is an agent that inhibits the formation of a blood clot. Examples of anticoagulants include, but are not limited to, specific inhibitors of thrombin, factor IXa, factor Xa, factor XIa, factor XIIa, or factor VIIa, heparin and derivatives, vitamin K antagonists, and anti-tissue factor antibodies. Examples of specific inhibitors of thrombin include hirudin, bivalirudin, Argatroban and lepirudin Examples of heparin and derivatives include unfractionated heparin (UFH), low molecular weight heparin (LMWH), such as enoxaparin Dalteparin Danaparin and synthetic pentasaccharides such as fondaparinux Examples of vitamin K antagonists include warfarin Phenocoumarol, acenocoumarol Chloroindanedione, dicoumarol, diphenylindanone, dicoumarol ethyl acetate, phenprocoumon, phenindanedione and ticlocoumarin. In one embodiment, the anticoagulant is an inhibitor of factor Xa. In one embodiment, the anticoagulant is betrixaban.

[0089] "Anticoagulant therapy" refers to a therapeutic regimen administered to a patient to prevent unwanted blood clots or thrombosis. Anticoagulant therapy includes the administration of one or a combination of two or more anticoagulants or other agents at doses and regimens suitable for treating or preventing unwanted blood clots or thrombosis in a patient.

[0090] The term "factor Xa inhibitor" or "inhibitor of factor Xa" refers to a compound that can directly or indirectly inhibit the activity of coagulation factor Xa in catalyzing the conversion of prothrombin to thrombin in vitro and / or in vivo.

[0091] "Direct factor Xa inhibitors" bind directly to fXa, and non-limiting examples include NAP-5, rNAPc2, tissue factor pathway inhibitor (TFPI), DX-DX-9065a (as described in, e.g., Herbert, JM, et al., J Pharmacol Exp Ther. 1996 276(3):1030-8), YM-60828 (as described in, e.g., Taniuchi, Y., et al., Thromb Haemost. 1998 79(3):543-8), YM-150 (as described in, e.g., Eriksson, BI et al., Blood 2005; 106(11), Abstract 1865), apixaban, rivaroxaban, TAK-442, PD-348292 (as described in, e.g., Pipeline Insight: Antithrombotics-Reaching the Untreated Prophylaxis Market, 2007), omixaban, edoxaban (such as described in, for example, Hylek EM, Curr Opin Invest Drugs 2007 8 (9): 778-783), LY517717 (such as described in, for example, Agnelli, G., et al., J. Thromb. Haemost. 2007 5 (4): 746-53), GSK913893, razaxaban, betrixaban or its pharmaceutically acceptable salt and combination thereof. In a specific aspect, direct factor Xa inhibitor is rivaroxaban. In some aspects, direct fXa inhibitor is a small molecule compound.

[0092] The inhibition of fXa activity by "indirect factor Xa inhibitors" is mediated by one or more other factors. Non-limiting examples of indirect factor Xa inhibitors include fondaparinux, edaparinux, biotinylated edaparinux, enoxaparin, fragmin, tinzaparin, low molecular weight heparin ("LMWH") and combinations thereof. In a specific aspect, the indirect factor Xa inhibitor is enoxaparin.

[0093] In one embodiment, the Factor Xa inhibitor is selected from betrixaban, rivaroxaban, LMWH, DX-9065a, YM-60828, YM-150, PD-348292, omixaban, edoxaban, LY517717, GSK913893, razoxaban, apixaban, and combinations thereof.

[0094] 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. Patents 6,376,515, 6,835,739 and 7,598,276, the contents of which are incorporated herein by reference. Betrixaban is known to be a specific inhibitor of factor Xa.

[0095] "Neutralize", "reverse" or "counteract" the activity of fXa inhibitors or similar phrases refer to inhibiting or blocking factor fXa inhibition or the anticoagulant function of fXa inhibitors. Such phrases refer to partially inhibiting or blocking function in vitro and / or in vivo as well as inhibiting or blocking most or all of the activity of fXa inhibitors.

[0096] "Effective amount" refers to the amount of a derivative sufficient to induce a desired biological result and / or therapeutic result. The result may be a sign, symptom or cause of disease alleviation, or any other desired change in a biological system. In the present disclosure, the result will generally involve one or more of the following: neutralizing the fXa inhibitor administered to the patient, reversing the anticoagulant activity of the fXa inhibitor, removing the fXa inhibitor from plasma, restoring hemostasis, and reducing or stopping bleeding. The effective amount will vary according to the specific antidote used, the specific fXa inhibitor administered to the subject, the dosing regimen of the fXa inhibitor, the administration time of the antidote, the subject and the disease condition treated, the subject's weight and age, the severity of the disease condition, the mode of administration, etc., all of which factors can be easily determined by one of ordinary skill in the art.

[0097] In certain aspects, the solution is applied to deliver an amount of about 10 milligrams (mg) to about 2 grams (g) of fXa antidote. Other amounts of the 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 aspects, the amount of the r- antidote used is about 400 mg or 960 mg. In some aspects, the amount of the r- antidote used is about 10 mg to about 100 mg; about 15 mg to about 95 mg; and about 20 mg to about 80 mg.

[0098] Upon administration, 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%.

[0099] Whether the method has been achieved, ie, inhibition or reversal of the Factor Xa inhibitor, can be determined by a number of in vitro assays, such as thrombin generation assays, and clinical coagulation assays, such as aPTT, PT, and ACT.

[0100] One aspect of the present disclosure relates to a method for selectively binding and inhibiting an exogenously administered fXa inhibitor in a subject receiving anticoagulant therapy with a fXa inhibitor, the method comprising administering to the subject a solution of an effective amount of a lyophilized formulation. Patients suitable for this therapy have previously received anticoagulant therapy, for example, they have received one or more anticoagulants, such as direct or indirect fXa inhibitors.

[0101] In another aspect, the methods provided herein selectively bind and inhibit an exogenously administered factor Xa inhibitor in a subject receiving anticoagulant therapy with a factor Xa inhibitor, the method comprising administering a solution of a lyophilized formulation to the subject. The subject may be a cell or a mammal, such as a human.

[0102] The subject of the administration of the lyophilized formulation of dissolution described herein and the attached method includes those subjects who are experiencing or are susceptible to clinical major bleeding events or clinically significant non-major bleeding events. The example of clinical major bleeding events is selected from the group consisting of: bleeding, bleeding in vital organs, bleeding requiring reoperation or new treatment procedures, and bleeding index ≥ 2.0 with relevant obvious bleeding. (Turpie AGG, et al., NEJM, 2001, 344: 619-625.) In addition, the subject may be experiencing or susceptible to non-major bleeding events, which is selected from the group consisting of: persistent or recurrent and a large amount of or epistaxis that will not stop in the absence of intervention, rectal or urinary tract bleeding that will not rise to the level of treatment procedures, injection sites that occur spontaneously or in the case of minor trauma, or significant hematomas in other places, more than the large amount of blood loss associated with the surgical procedure that does not require drainage, and bleeding that requires unplanned blood transfusion.

[0103] In some embodiments, the dissolved lyophilized formulation is administered following an overdose of the fXa inhibitor or prior to a surgery that may expose the subject to a risk of bleeding.

[0104] Example

[0105] The following examples are included to demonstrate specific embodiments of the present disclosure. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that work well in the practice of the present disclosure, and therefore can be considered to constitute preferred modes for the practice of the present invention. However, in accordance with the present disclosure, it will be appreciated by those skilled in the art that many changes may be made in the specified embodiments disclosed and still obtaining similar or similar results without departing from the spirit and scope of the present disclosure.

[0106] Example 1: Purification of r-Antidote from Cell Culture

[0107] This example demonstrates the development of culture conditions and downstream purification processes for the production of r-Antidote. A commercial culture medium tailored for CHO cells was tested against CHO cells transfected with a construct containing a nucleic acid sequence (SEQ ID NO:7) encoding an r-Antidote precursor (SEQ ID NO:2) that generates a double-stranded r-Antidote (SEQ ID NO:3) upon removal of the -RKRRKR- linker (SEQ ID NO:6). Under appropriate conditions, ProCHO TM The culture medium is capable of producing titers as high as 75-95 mg / L.

[0108] A downstream purification process ("downstream process") was developed to purify the antidote from cultured cells. In some initial development runs, various membranes and columns were tested.

[0109] In two test runs, it was found that the clarified harvest contaminated the membrane when the product was concentrated by ultrafiltration (UF), or contaminated the chromatographic medium when the product was concentrated by the first chromatography step while bypassing UF. Experiments were performed to determine the cause of the contamination and to determine the steps to eliminate the contamination, because it was believed that the concentrated product was needed in the early stages of the process. It was subsequently determined that the contamination was caused by high molecular weight (HMW) protein impurities secreted into the culture medium during cell culture. This HMW protein was only partially dissolved in the clarified harvest, and when the concentrate became increasingly insoluble, it caused unacceptable contamination of the UF membrane or chromatographic column.

[0110] After evaluating several depth filters, ion exchange membranes, and chromatography media, it was found that one particular depth filter (Millipore TMX0HC grade filter) can remove most impurities while obtaining acceptable product yields. The product can then be concentrated up to 10 times by UF. This filter replaced the original filter in the clarification process and two additional runs were implemented.

[0111] Figure 1 The purification process 100 resulting from this development work is shown. After the cells are harvested (step 101) and purified using Millipore TM After the XOHC filter is clarified to remove HWM impurities, the sample is concentrated 10 times (step 102). Regenerated cellulose can be used for concentration. In step 103, virus inactivation (using detergent / solvent; final concentration: 1% Triton X-100, 0.3% tributyl phosphate) can be performed to inactivate the enveloped virus in the cell culture. After removing the virus, 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 antidotes. In step 107, hydrophobic interaction resins can be used to further remove remaining host cell proteins. Optionally, after these purification steps, a final virus removal filtration step can be used to remove any remaining viruses.

[0112] This process is able to recover approximately 30-35% of the antidote expressed in the cell culture, resulting in the production of approximately 22-33 mg of antidote protein per liter of cell culture.

[0113] Example 2: Scale-up of manufacturing process

[0114] Based on the process shown in Example 1, an improved process was developed for the purpose of scale-up.

[0115] The medium of Example 1 was able to produce a titer of about 75-95 mg / L. To increase production, many other CHO media were tested. One of them showed a 2-3 fold increase in cell growth and peak cell density (titer = 200-225 mg / L) and could also be scaled up to 10,000 L.

[0116] Affinity chromatography was developed for the bulk extraction of antidotes. Affinity ligands included Kunitz trypsin inhibitor (21 kDa; pI = 4.5) extracted from whole soybeans or flour. Soybean trypsin inhibitor (STI) formed a 1:1 complex with the antidote. The scalability and compatibility of noncompetitive inhibitor elution conditions were identified, which included 25 mM sodium acetate, 1 M arginine, pH 5.2.

[0117] In the process developed in Example 1, a harvest clarification step using a depth filter was used to remove HMW impurities, followed by a 10-fold concentration. In addition, a viral inactivation step used a detergent (Triton X-100) and a solvent (tributyl phosphate). These steps were evaluated for suitability and efficiency for affinity capture.

[0118] The data show that depth filtration is not required, and the use of solvent and 10x concentration steps reduces the recovery rate of the product. Therefore, in further development, depth filtration and concentration steps are eliminated, and the virus inactivation uses only detergent (TritonX-100), without solvent. Virus inactivation is achieved by adding 10% Triton X-100 (final concentration: 0.5% Triton X-100) in a ratio of 52.7ml buffer per liter of product. This discovery is unexpected because solvents are often used for sample preparation before being loaded into affinity columns. It should also be noted that when harvesting cell cultures, a centrifugal step is used to remove precipitate.

[0119] After 15 minutes of mixing time at room temperature and a hold time of at least 1 hour, the treated product was filtered through a filter train consisting of a Sartogrud NF filter (0.8 / 0.2 μm) prefilter over a Sartopore 2 (0.45 / 0.22 μm) prefilter into a 500 L vessel with an integrated 0.22 μm filter, with each column load filtered on the day of use.

[0120] The product was eluted from the STI affinity resin using an elution buffer containing arginine (25 mM sodium acetate / 1.0 M arginine, pH 5.2).

[0121] After affinity capture, some polishing steps were used to remove remaining impurities, which included DNA, host cell proteins (HCPs), and the leachate of affinity ligands. Two chromatographic options were tested, including cation exchange and mixed-mode ion exchange. However, there was no process-specific host cell protein assay available for characterization. Therefore, commercial ELISA used as an alternative and LC-MS / MS quantification of specific HCPs were used as supplementary characterization. In addition, 2D silver staining evaluation was used for qualitative characterization.

[0122] Figure 3 Evaluation of three potential downstream processes (DSPs) is shown. Three DSPs were identified based on the clearance of the affinity ligand leachate. To evaluate product quality and process-related impurity clearance, the same affinity eluate pool was forward processed on each stream and compared for product recovery and CHO host cell protein clearance (HCP; expressed in parts per million (ppm)).

[0123] The product quality of the eluate from each DSP process was also tested (table below).

[0124]

[0125] Table Notes: Product quality evaluation of the three downstream processes evaluated. Eluates from three DSPs were evaluated ( Figure 3 ) product quality. No effect on charge-based heterogeneity (IEX) or reverse phase (RP) was found. However, samples from DSP-2 purified by the cation exchange polishing step showed an increase in the percentage of high molecular weight aggregates by size exclusion (SEC).

[0126] Based on the described tests, a scaled-up downstream process was developed, e.g. Figure 2 This process is faster than Figure 1 The process shown is simpler. However, the yield is 2-fold higher. Combined with a new cell culture system that increases protein yield 2-3 fold, the method described in Example 2 enables a 4- to 6-fold increase in overall antidote yield and enables a production scale of at least 10,000 L.

[0127] exist Figure 2 In the downstream process 200, after harvesting (201), a viral inactivation step (202) is used, which uses only detergent (Triton X-100) and no solvent. In the affinity capture step 203, a resin with an STI ligand is used, and the antidote is eluted with an elution buffer containing arginine (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.

[0128] At step 204, an anionic membrane is used to remove certain impurities (e.g., DNA, HCP) from the sample. An exemplary anionic membrane tested was the Sartobind Q membrane, which exhibited no deleterious effects on the detoxifier.

[0129] At step 205, an example type of mixed mode ion exchange is IEX mixed mode using CHT (ceramic hydroxyapatite). An example elution buffer is phosphate buffer. This step can be used to remove STI leachate.

[0130] At step 206, hydrophobic separation can be achieved using octyl sepharose chromatography as an example, and the protein can be eluted with a buffer containing NaCl. This step can be used to remove remaining HCPs.

[0131] The table below shows the purification and recovery yields from several test runs.

[0132]

[0133] Example 3: Characterization of produced proteins

[0134] In this example, methods were developed for characterizing the protein products produced by the methods developed above. The methods tested included isoelectric focusing (IEF), reduced reverse phase (Red-RP) and reduced peptide mapping (PMAP).

[0135] Isoelectric focusing is used to determine the charge heterogeneity of detoxification proteins. Isoelectric focusing is an electrophoresis technique used to separate proteins based on the isoelectric point (pI) of the protein or the pH when the protein has no net charge. Isoelectric focusing is performed using gels and reagents of pH 3-10 according to the instructions for electrophoresis (Novex IEF gel) of the Life Technologies manufacturer and staining with a colloidal blue staining kit (Invitrogen). The results are compared with the Serva 3-10 pI marker.

[0136] The test batch was diluted to 1.6 mg / mL in water and then diluted to 1:1 in IEF pH 3 10 sample buffer. Each sample was loaded at 10 μL (8.0 μg) and electrophoresis was performed at 100 V for 1 hour, then at 200 V for 1 hour, and at 500 V for 30 minutes. The gel was fixed in fixing solution (Sigma-Aldrich) for 30 minutes, stained in colloidal blue stain for 1 hour, and destained overnight in water.

[0137] Reducing reverse phase is an HPLC method that uses reverse phase chromatography to detect full-length and truncated forms of proteins. The sample is buffer exchanged into 6M guanidine hydrochloride / 50mM Tris pH 7.5 and reduced with DTT, then incubated at 50°C for 30 minutes. The sample is then alkylated in the dark using vinyl pyridine at room temperature for 90 minutes, followed by quenching with 1M DTT. The method utilizes an Agilent Zorbax C18 HPLC column to combine proteins with a 15% to 95% gradient of a stationary phase and a decreasing polarity mobile phase (0.1% trifluoroacetic acid in acetonitrile) to separate proteins based on their hydrophobicity. The UV wavelength is 214nm.

[0138] Peptide mapping of Lys-C digests using UPLC-UV / MS of Lys-C digests E Analysis. This method can provide more than 98% sequence coverage and characterize post-translational modifications, such as glycosylation, aspartate hydroxylation, and C-terminal lysine truncation. This method can also monitor stability indicators, such as asparagine deamidation and methionine oxidation. The sample batches are each reduced, alkylated, and treated with lysyl endopeptidase (Lys-C). The peptides produced by enzymatic digestion are separated by RP-UPLC and analyzed by mass spectrometry.

[0139] The table below shows the types of protein isoforms detected from the purified protein products, their percentages and sequences (Batch #1: downstream process developed in Example 1; Batch #2: downstream process developed in Example 2). It is interesting to note that the process of Example 2 significantly reduced the percentage of SEQ ID NO: 9 (from 9.0% to 2.7%) compared to the process of Example 1, which may be due to increased purification efficiency.

[0140] Light Chain Isotype:

[0141] Batch #1 Batch #2 Complete light chain (SEQ ID NO:4) 46.8％ 37.9％ Modified or truncated light chain 53.2％ 62.1％

[0142] Heavy Chain Isotype:

[0143]

[0144]

[0145] Example 4: Validation of the scale-up process

[0146] This example describes a process based on the process shown in Examples 1 and 2 for further verification.

[0147] Virus inactivation by detergents

[0148] The viral inactivation step was performed by adding 10% Triton X-100 to the harvested protein. The harvested protein corresponding to 1 Capto STI cycle (described below) was filtered from the harvest tank through a 4x30" Sartoguard NF filter used as a prefilter into a 10% Triton X-100 feed tank and then filtered through a 3x20" Sartoguard NF filter 0.8 / 0.2 μm. After the addition of 10% Triton X-100, the pool was mixed and then transferred to a holding tank through a 3x30" Sartoguard NF 0.8 / 0.2 μm filter. Once the holding time was completed, the inactivated pool was loaded onto the Capto STI column through a 6x30" Sartoguard NF filter 0.8 / 0.2 μm filter in series with a 3x30" Sartopore 2 0.45 / 0.2 μm filter. This sequence was repeated until the 4 cycles to be completed during the Capto STI operation were completed. After loading the second cycle, the 6x30" Sartoguard NF 0.8 / 0.2 μm filter was replaced.

[0149] Capto STI Chromatography

[0150] Capto STI resin is used to perform affinity column chromatography at ambient temperature. During the loading phase, product is bound to the resin, and pollutants flow through simultaneously. Product is recovered by destroying the interaction of protein and high concentration of arginine in elution buffer. Before use, the post is rinsed with water for injection (WFI), cleaned with 100mM phosphoric acid pH 3.0, and rinsed with WFI. The post is initially pre-balanced with pre-equilibrium buffer (20mM Tris-HCl, pH 7.4), then balanced with equilibrium buffer (20mMTris-HCl, 200mM NaCl, pH 7.4), and once the pH and conductivity of the post are within the specification range, the clarified harvest (HCCF) processed by Triton-X 100 is loaded. Each circulation of HCCF material is directly transferred to the post from its virus inactivation holding tank.

[0151] The column is washed with a balance buffer after loading, and eluted with a high arginine low pH elution buffer (elution buffer: 25mM sodium acetate, 1.0M arginine, pH 5.2). At the end of the cycle and before the next cycle, the column is rinsed with WFI, cleaned with 100mM phosphoric acid pH 3.0 again, and rinsed with WFI again. All eluents from several cycles are merged together. After collecting the last cycle, buffer tracking is performed, bypassing the column, so that the product of the wash-out is rinsed into the collection container from the pipeline downstream of the column. After all cycles are completed, the column is rinsed with WFI, cleaned with 100mM phosphoric acid pH 3.0, rinsed with WFI, neutralized with pre-equilibrium and balance buffer and stored in an ethanol storage solution.

[0152] The Capto STI eluate pool was mixed with 1.0 M arginine hydrochloride pH 7.0 to a final concentration of 100 mM arginine hydrochloride to stabilize the pool. The resulting pool was then concentrated and diafiltered on a 10 KDa Ultracel Pellicon 3 membrane to remove most of the arginine, allowing loading into subsequent chromatography steps.

[0153] Sartobind Q Membrane Chromatography

[0154] Anion exchange membrane chromatography was performed at ambient temperature using Sartobind Q Jumbo cartridges. During the loading phase, the product flows through the membrane while contaminants bind. Prior to use, the cartridge was flushed with equilibration buffer to remove storage wetting agents. The cartridge was rinsed with 0.5M NaOH. The cartridge was then equilibrated with equilibration buffer and once the pH and conductivity of the column were within specification, the concentrated diafiltered Capto STI eluate was loaded. A loading material cycle 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 around the cartridge to flush the product from the line downstream of the column into the collection vessel.

[0155] Ceramic Hydroxyapatite Chromatography (CHT)

[0156] Mixed mode column chromatography was performed at ambient temperature using CHT I type 40 μm resin. During the loading phase, the product binds to the resin while some contaminants flow through. The product was recovered by increasing the sodium concentration in a linear gradient. Prior to use, the column was cleaned with 1.0 M NaOH and rinsed with a small amount of equilibration buffer. The column was initially pre-equilibrated with pre-equilibration buffer, then equilibrated with equilibration buffer, and once the pH and conductivity of the column were within specification, Sartobind Q eluent was loaded.

[0157] Each cycle loaded was transferred directly to the column from its collection tank. After loading, the column was washed with equilibration buffer. A gradient 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) was then run.

[0158] At the end of the cycle and before the next cycle, the post is rinsed with equilibration buffer, then stripped with high phosphate post-elution wash buffer, rinsed with equilibration buffer, cleaned again with 1.0M NaOH, and rinsed again with equilibration buffer. All eluents from several cycles are combined together. After the last cycle is collected, buffer tracking is performed, bypassing the post so that the eluted product is rinsed from the line downstream of the post into a collection container. After all cycles are completed, the post is rinsed with equilibration buffer, cleaned with 1.0M NaOH, and stored in a caustic / phosphate storage solution.

[0159] Octyl Sepharose 4FF Chromatography

[0160] Octyl Sepharose FF resin was used to perform hydrophobic interaction chromatography at ambient temperature. During the loading phase, the product flowed through the resin while the contaminants were bound. Before use, the post was cleaned with 1.0M NaOH. The post was initially pre-equilibrated with WFI, then equilibrated with equilibrium buffer, and once the pH and conductivity of the post were within the specification range, the CHT eluent was loaded. Each cycle loaded was directly transferred to the post from its collection tank. After loading, the post was washed with equilibrium buffer. At the end of the cycle and before the next cycle, the post was stripped with WFI and cleaned with 1.0M NaOH again. After collecting the last cycle, buffer tracking was performed, bypassing the post so that the product was flushed into the collection container from the line downstream of the cartridge. After all cycles were completed, the post was stripped with WFI, cleaned with 1.0M NaOH, and stored in a caustic storage solution.

[0161] Planova 20N Virus Reduction Filtration (VRF)

[0162] Virus reduction filtration was performed on a Planova 20N virus reduction filter. The filter was operated in terminal mode, with the product passing through the filter while any remaining viral particles were retained in the fibers. Prior to use, the filter was rinsed with Octyl Sepharose equilibration buffer. The product was then passed through the filter and then rinsed with additional equilibration buffer. The filter was tested for integrity and discarded after use.

[0163] 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 recovered.

[0164] ***

[0165] Unless defined otherwise, 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.

[0166] The present invention illustratively described herein can be properly practiced in the absence of any one or more elements, one or more limitations not specifically disclosed herein. Thus, for example, the terms "comprises," "includes," "containing," and the like should be interpreted expansively and without limitation. In addition, the terms and expressions employed herein have been used as descriptive terms rather than limiting terms, and there is no intention to use these terms and expressions to exclude any equivalents or portions thereof of the features shown or described, but it is recognized that various modifications within the scope of the claimed invention are possible.

[0167] Therefore, it should be understood that although the present invention is specifically disclosed by preferred embodiments and optional features, those skilled in the art may modify, improve and change the invention embodied in the text, and such modifications, improvements and changes are considered to be within the scope of the present invention. The materials, methods and examples provided herein are representative of preferred embodiments, are exemplary, and are not intended to be used as limitations on the scope of the present invention.

[0168] The invention has been 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 proviso or negative limitation to remove any subject matter from the generic regardless of whether the deleted material is specifically described herein.

[0169] In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0170] All publications, patent applications, patents, and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each reference was individually incorporated by reference. In the event of a conflict, the present specification, including definitions, will control.

[0171] It should be understood that although the present disclosure has been described in conjunction with the above embodiments, the above description and examples are intended to illustrate rather than limit 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 the present disclosure belongs.

Claims

1. A method for preparing a polypeptide product expressed by a polynucleotide construct, wherein the polynucleotide construct comprises the nucleic acid sequence of SEQ ID NO: 7, the method comprising: adding a detergent to a sample containing a polypeptide product expressed by the polynucleotide construct; loading the sample into a soybean trypsin inhibitor-based affinity chromatography and eluting the polypeptide with a first elution buffer to produce a first eluted sample, wherein the loaded sample does not contain an organic solvent; loading the first elution sample into a ceramic hydroxyapatite type I chromatograph, and eluting the polypeptide with a second elution buffer comprising at least 1 M inorganic salt to produce a second elution sample; and loading the second elution sample onto an Octyl Sepharose chromatograph and eluting the polypeptide with a third elution buffer comprising at least 2 mM sodium chloride, A purified sample comprising the polypeptide product is thereby prepared.

2. The method of claim 1, wherein the second elution buffer comprises at least 2 M of an inorganic salt.

3. The method of claim 2, wherein the inorganic salt is sodium chloride.

4. The method of claim 1, wherein: i) the detergent comprises Triton X-100 (polyethylene glycol p-(1,1,3,3-tetramethylbutyl)-phenyl ether); and / or ii) The first elution buffer contains 0.5 M to 2 M arginine. The method of claim 4 , wherein the first elution buffer has a pH of 5 to 5.

4.

6. The method of claim 1, further comprising: i) a purification step with anion exchange chromatography, wherein the anion exchange chromatography is applied after the affinity chromatography; and / or ii) filtering one or more of said samples with a nano fishnet membrane filter, wherein said filtering with said nano fishnet membrane filter is performed before loading said sample onto said soybean trypsin inhibitor-based affinity chromatography.

7. The method of claim 1, wherein the purified sample contains less than 1% of contaminating proteins not expressed by the polynucleotide construct.

8. The method of claim 1, wherein the polypeptide product is expressed in a cell containing the polynucleotide construct.

9. The method of any preceding claim, wherein the polypeptide product is a two-chain polypeptide comprising a light chain and a heavy chain.

10. The method of claim 9, wherein: i) 20% to 50% of the polypeptide products in the purified sample have: (A) a heavy chain consisting of the amino acid sequence of SEQ ID NO:5; or (B) a heavy chain consisting of the amino acid sequence of SEQ ID NO:5; and 5%-95% of the heavy chain consisting of the amino acid sequence of SEQ ID NO:5 has two O-linked glycosylations, and 5%-95% of the heavy chain consisting of the amino acid sequence of SEQ ID NO:5 has one O-linked glycosylation; or ii) 40-80% of the polypeptide products in the purified sample have: (A) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8; or (B) a heavy chain consisting of the amino acid sequence of SEQ ID NO: 8; and at least 90% of the heavy chain consisting of the amino acid sequence of SEQ ID NO: 8 has one O-linked glycosylation; or iii) 2%-12% of the polypeptide products in the purified sample have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 9; or iv) 0.1%-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; or v) 2%-8% of the polypeptide products in the purified sample have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 11; or vi) 35%-60% of the polypeptide products in the purified sample have a light chain consisting of the amino acid sequence of SEQ ID NO:

4.

11. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polypeptide portion of a double-chain polypeptide expressed in a cell containing a polynucleotide construct comprising a nucleic acid sequence of SEQ ID NO: 7, wherein: 35%-60% of the double-chain polypeptides have a light chain consisting of the amino acid sequence of SEQ ID NO: 4; 20%-60% of the double-chain polypeptides have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 5; 40%-60% of the double-chain polypeptide has 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.

12. The pharmaceutical composition according to claim 11, wherein: i) less than 5% or less than 3% of the double-chain polypeptides have a heavy chain consisting of the amino acid sequence of SEQ ID NO: 9; and / or ii) 0.1%-1.5% of the double-chain polypeptide has a heavy chain consisting of the amino acid sequence of SEQ ID NO: 10; and / or iii) 2%-8% of the double-chain polypeptides have a heavy chain consisting of the amino acid sequence of SEQ ID NO:

11.

13. The pharmaceutical composition of claim 11, wherein: i) 30%-70% of the heavy chains consisting of the amino acid sequence of SEQ ID NO: 5 have two O-linked glycosylation; and / or ii) 30%-70% of the heavy chains consisting of the amino acid sequence of SEQ ID NO: 5 have one O-linked glycosylation; and / or iii) at least 90% of said heavy chains consisting of the amino acid sequence of SEQ ID NO: 8 have one O-linked glycosylation.

14. The pharmaceutical composition of claim 11, wherein: i) the pharmaceutical composition is lyophilized; and / or ii) the pharmaceutical composition further comprises L-arginine hydrochloride or L-arginine acetate; and / or iii) the pharmaceutical composition further comprises sucrose; and / or iv) the pharmaceutical composition further comprises mannitol.

15. Use of the pharmaceutical composition of any one of claims 11 to 14 for the preparation of a medicament for reversing or inhibiting anticoagulation in a patient undergoing anticoagulation therapy with a factor Xa inhibitor.

16. A purified sample comprising a polypeptide product expressed by a polynucleotide construct, wherein the polynucleotide construct includes the nucleic acid sequence of SEQ ID NO:7, and the polypeptide product is a double-chain polypeptide comprising a light chain and a heavy chain, wherein about 2%-12% of the polypeptide products in the purified sample have a heavy chain consisting of the amino acid sequence of SEQ ID NO:9.

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