Separation of VWF and VWF propeptide by chromatographic methods.

Abstract

The present invention relates to a method for separating mature von Willebrand factor (mat-VWF) from von Willebrand factor propeptide (VWF-PP) by incubating a composition, the method comprising inducing dissociation of mat-VWF and VWF-PP by disrupting non-covalently associated mat-VWF and VWF-PP, wherein the dissociation is induced by (i) adding at least one chelating agent or (ii) increasing the pH to at least pH 7, and then recovering the mat-VWF to obtain highly purified propeptide-depleted mature VWF (mat-VWF).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 646,109, filed March 21, 2018, which is incorporated herein by reference in its entirety.

[0002] FIELD OF THE INVENTION The present invention relates to a method for separating mature von Willebrand factor (VWF) from von Willebrand factor propeptide (VWF-PP). [Background technology]

[0003] Background of the Invention During protein maturation in cells, proteins undergo post-translational modifications to mature. These modifications include acetylation, methylation, glycosylation, and proteolytic cleavage, among others. These modifications are often necessary for protein function and activity and can also affect the efficiency of proteins, especially enzymes.

[0004] A proprotein or protein precursor is an inactive protein that is converted to an active form by one or more of these post-translational modifications, particularly by cleavage of the propeptide from the proprotein.

[0005] Active forms of these proteins can be useful therapeutic and / or diagnostic proteins. However, active proteins are usually available in vivo in very small amounts. Therefore, active proteins are recombinantly produced from their proproteins, which are preferably activated in vitro by contacting them with a recombinant activating enzyme (e.g., a protease).

[0006] Von Willebrand factor (VWF) is a glycoprotein that circulates in plasma as a series of multimers ranging in size from approximately 500 to 20,000 kD. The full-length VWF cDNA has been cloned; the propolypeptide corresponds to amino acid residues 23 to 764 of the full-length preproVWF (Eikenboom et al. (1995) Haemophilia, 1, 77-90). The multimeric form of VWF consists of 250 kD polypeptide subunits linked to each other by disulfide bonds. VWF mediates initial platelet adhesion to the subendothelium of injured vascular walls, and larger multimers exhibit enhanced hemostatic activity. Multimerized VWF binds to the platelet surface glycoprotein Gp1bα through interactions at the A1 domain of VWF, promoting platelet adhesion. Other sites on VWF mediate binding to the vascular wall. Thus, VWF forms a bridge between platelets and the vessel wall, which is essential for platelet adhesion and primary hemostasis under conditions of high shear stress. Normally, endothelial cells secrete high molecular weight forms of VWF, and low molecular weight VWF forms result from proteolytic cleavage. Ultra-high molecular weight multimers are stored in Weibel-Palade bodies of endothelial cells and are released upon stimulation by agonists such as thrombin and histamine.

[0007] In industrial applications, VWF, particularly recombinant VWF (rVWF), is synthesized and expressed together with rFVIII in genetically modified cell lines, such as genetically modified CHO cell lines. The function of the coexpressed rVWF is to stabilize rFVIII during the cell culture process. rVWF is synthesized intracellularly as a prepropeptide VWF (prepro-VWF), which contains a high molecular weight propeptide (VWF-PP) attached to the N-terminus of the mature VWF (matVWF) subunit. Upon maturation in the endoplasmic reticulum and Golgi apparatus, VWF-PP is cleaved by the action of the intracellular protease furin and secreted as a homopolymer of identical subunits consisting of a dimer of the expressed protein. In some cases, furin cleavage generates a heterodimeric complex containing mature VWF noncovalently bound to the VWF propeptide.

[0008] VWF-PP can be separated from mature VWF by in vitro treatment with furin or furin-like proteases (Schlokat U. et al. (1996) Biotechnol. Appl. Biochem. 24:257-267 (Non-Patent Document 2); Preininger A. et al. (1999) Cytotechnology 30:1-15 (Non-Patent Document 3)). Furin belongs to the family of proprotein convertases and is a Ca 2+ The enzyme specifically cleaves the C-terminal peptide bond of arginines within a specific sequence, including arginines at positions -1 and -4. This sequence is found in many human proteins, indicating that furin plays a key role in the maturation of many human propeptide-proteins. The furin used in the methods of the present invention is preferably of recombinant origin. Recombinantly produced proteases are useful because they can be produced in large quantities. In some embodiments, furin is obtained from crude cell culture supernatants of cell lines secreting the proteases or cell extracts.

[0009] Current conventional methods involve either incubating prepropeptide VWF with a protease in the liquid phase, allowing maturation (e.g., cleavage of the propeptide from the proprotein) to occur in free solution and unbound, or, as described, for example, in WO 2000 / 049047, immobilizing the protease on a solid support and contacting and incubating it with a preparation containing VWF-PP to generate mature VWF (see, for example, WO 2000 / 049047). VWF is synthesized by endothelial cells and megakaryocytes as a prepropeptide VWF ("prepro-VWF"), which consists largely of a repeat domain. After cleavage of the signal peptide, prepro-VWF dimerizes via disulfide bonds in the carboxy-terminal region of the endoplasmic reticulum. Additional disulfide bonds are formed near the amino termini of the subunits, resulting in the formation of multimers in the Golgi apparatus. Following assembly into multimers, proteolytic cleavage of the VWF propeptide occurs by the propeptide-processing protease furin. After cleavage, the VWF propeptide remains noncovalently associated with VWF multimers to form mature VWF / VWF-PP complexes. Upon stimulation, the complexes are secreted into the blood, and the VWF propeptide dissociates from the VWF multimers. Therapeutically effective mature VWF multimers can be produced by recombinantly expressing pro-VWF in mammalian cell lines and processing the pro-VWF protein to mature VWF through a series of in vitro cleavage and purification steps. However, there remains a need in the art to produce highly pure, therapeutically effective mature VWF multimer preparations (mat-rVWF), and the present invention fulfills this need by providing a method for obtaining highly pure mat-rVWF preparations, which includes, for example, adding a chelating agent and / or increasing the pH to at least pH 7 during the purification process after furin maturation to facilitate separation of VWF-propeptide from mat-rVWF. [Prior art documents] [Patent documents]

[0010] [Patent Document 1] WO2000 / 049047 [Non-patent literature]

[0011] [Non-Patent Document 1] Eikenboom et al (1995) Haemophilia, 1, 77-90 [Non-patent document 2] Schlokat U. et al. (1996)Biotechnol.Appl.Biochem.24:257-267 [Non-patent document 3] Preininger A. et al. (1999) Cytotechnology 30:1-15 Summary of the Invention

[0012] The present invention provides a method for obtaining a composition comprising highly purified mature recombinant rVWF (mat-rVWF) depleted of propeptides, said method comprising the steps of: a) providing a solution containing mat-rVWF / rVWF-PP complex, mat-rVWF, and rVWF propeptide (rVWF-PP); b) inducing dissociation of the mat-rVWF / rVWF-PP complex in the solution of a) into mat-rVWF and rVWF-PP, wherein the dissociation occurs by disrupting the non-covalently associated mat-rVWF and rVWF-PP, and the dissociation is i. adding at least one chelating agent, or ii. Raise the pH to at least pH 7 inducing by; and c) recovering the mat-rVWF to obtain a highly purified mat-rVWF composition containing at least 95% mature rVWF and less than 5% rVWF-PP.

[0013] In some embodiments, the highly pure mat-rVWF composition comprises at least 96% mat-rVWF and less than 4% rVWF-PP, at least 97% mat-rVWF and less than 3% rVWF-PP, at least 98% mat-rVWF and less than 2% rVWF-PP, at least 99% mat-rVWF and less than 1% rVWF-PP, or at least 99.5% mat-rVWF and less than 0.5% rVWF-PP, or 99.9% mat-rVWF and less than 0.1% rVWF-PP.

[0014] In some embodiments, the solution is selected from the group consisting of cell culture medium, antibody column flow-through solution, and buffer solution.

[0015] In some embodiments, the solution is treated with furin prior to step a).

[0016] In some embodiments, the solution is an antibody column flow-through solution.

[0017] In some embodiments, at least one chelator is a divalent cation chelator, hi some embodiments, the divalent cation chelator is selected from the group consisting of EDTA, EGTA, CDTA, and citrate.

[0018] In some embodiments, the pH is increased to at least 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0. In some embodiments, the pH is increased to at least about 7.2 to about 7.8. In some embodiments, the pH is increased to at least about 7.6. In some embodiments, the pH is increased by the addition of a basic amino acid, Tris, NaOH, tricine, or ethanolamine.

[0019] In some embodiments, the recovering in step b) of the methods described herein includes one or more protein separation methods. In some embodiments, the one or more protein separation methods are selected from the group consisting of ion exchange chromatography (IEC), size exclusion chromatography (SEC), physical size separation by membrane technology, and affinity chromatography. In some embodiments, the protein separation method is size exclusion chromatography (SEC). In some embodiments, the one or more protein separation methods is ion exchange chromatography (IEC). In some embodiments, the ion exchange chromatography (IEC) is cation exchange chromatography. In some embodiments, the ion exchange chromatography (IEC) is a combination of anion exchange chromatography and cation exchange chromatography.

[0020] In some embodiments, the one or more protein separation methods include a buffer system, wherein the buffer system comprises one or more buffers. In some embodiments, the one or more buffers include a wash buffer, wherein the one or more wash buffers include one, two, three, four, and / or five wash buffers, and where the one or more buffers include five wash buffers, the first, second, third, and / or fifth wash buffer has a higher pH than the fourth wash buffer, and where the one or more buffers include four wash buffers, the first, second, and / or fourth wash buffer has a higher pH than the third wash buffer. In some embodiments, the method further includes a virus inactivation step after the first wash buffer, optionally with a higher pH than the third and / or fourth wash buffers. In some embodiments, the one or more buffers include the one or more chelating agents. In some embodiments, the one or more buffers exhibit a pH of at least 7.

[0021] In some embodiments, the one or more protein separation methods include a buffer system, wherein the buffer system includes one or more loading buffers. In some embodiments, the one or more loading buffers include the one or more chelating agents. In some embodiments, the one or more loading buffers exhibit a pH of at least 7.

[0022] In some embodiments, the one or more protein separation methods include a buffer system, wherein the buffer system includes one or more loading buffers, wash buffers, and / or elution buffers. In some embodiments, the one or more loading buffers, wash buffers, and / or elution buffers include the one or more chelating agents. In some embodiments, the one or more loading buffers, wash buffers, and / or elution buffers exhibit a pH of at least 7. In some embodiments, the one or more loading buffers, wash buffers, and / or elution buffers include the one or more chelating agents and exhibit a pH of at least 7.

[0023] In some embodiments, the buffer system is selected from the group consisting of glycine HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TrisHCl (tris(hydroxymethyl)-aminomethane), histidine, imidazole, acetate citrate, MES, and 2-(N-morpholino)ethanesulfonic acid.

[0024] In some embodiments, the buffer further comprises one or more monovalent cations. In some embodiments, the one or more monovalent cations are selected from the group consisting of Na+, K+, Li+, and Cs+. In some embodiments, the monovalent cation is Na+.

[0025] In some embodiments, the buffer further comprises one or more monovalent, divalent, and / or trivalent anions. In some embodiments, the one or more monovalent, divalent, and / or trivalent anions are Cl. - , acetic acid - , SO4 2- , Br - , and citric acid3- is selected from the group consisting of:

[0026] In some embodiments, the buffer system comprises at least one buffer exhibiting a conductivity of ≧0.5 mS / cm at 25° C. In some embodiments, the buffer system comprises at least one buffer exhibiting a conductivity of 15.0±0.2 mS / cm at 25° C.

[0027] In some embodiments, the buffer further comprises one or more non-ionic detergents, hi some embodiments, the non-ionic detergents are selected from the group consisting of Triton X-100, Tween 80, and Tween 20.

[0028] In some embodiments, the buffer further comprises one or more additional substances selected from the group consisting of non-reducing sugars, sugar alcohols, and polyols.

[0029] In some embodiments, the highly pure mat-rVWF composition has a host cell (HC) impurity level of ≦2.0%. In some embodiments, the highly pure mat-rVWF composition has a host cell (HC) impurity level of ≦0.6%.

[0030] In some embodiments, the solution comprising mat-rVWF / rVWF-PP complexes, mat-rVWF, and rVWF-PP is obtained from a capture step of rVWF.

[0031] In some embodiments, the solution comprising mat-rVWF / rVWF-PP complexes, mat-rVWF, and rVWF-PP is derived from a process comprising a FVIII immunoaffinity step and an anion exchange chromatography step.

[0032] The present invention also provides a method for obtaining a composition comprising highly purified mature recombinant rVWF depleted of propeptides (highly purified mat-rVWF), said method comprising the steps of: a) loading a solution containing pro-rVWF, mat-rVWF / rVWF-PP complex, mat-rVWF, and / or rVWF propeptide (rVWF-PP) onto an anion exchange column, and allowing the pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF to bind to the anion exchange column; b) washing the anion exchange column of a) containing the bound pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF with one or more wash buffers; c) treating the column of b) containing the bound pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF with furin, wherein the furin cleaves the pro-rVWF into mat-rVWF and rVWF-PP; d) eluting the bound pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF from the column of c) with an elution buffer, wherein the elution buffer induces dissociation of the rVWF-PP from mat-rVWF non-covalently associated with the rVWF-PP, and the dissociation is i. adding at least one chelating agent to said elution buffer, or ii. increasing the pH of the elution buffer to at least pH 7. inducing by; and e) Separating and recovering the mat-rVWF from the rVWF-PP to obtain a highly purified mat-rVWF composition, wherein the highly purified mat-rVWF composition contains at least 95% mature rVWF and less than 5% rVWF-PP.

[0033] In some embodiments, a) and b) occur simultaneously in a single step.

[0034] In some embodiments, the one or more buffers comprise a wash buffer, and the one or more wash buffers comprise 1, 2, 3, 4, and / or 5 wash buffers, where if the one or more buffers comprise 5 wash buffers, the first, second, third, and / or fifth wash buffer have a higher pH than the fourth wash buffer, and where if the one or more buffers comprise 4 wash buffers, the first, second, and / or fourth wash buffer have a higher pH than the third wash buffer. In some embodiments, the method further comprises a virus inactivation step after the first wash buffer, optionally at a higher pH than the third and / or fourth wash buffers.

[0035] In some embodiments, the solution in a) comprises flow-through from a monoclonal antibody column, wherein the monoclonal antibody is a FVIII monoclonal antibody.

[0036] In some embodiments, the solution in a) is selected from the group consisting of cell culture medium, antibody column flow-through solution, and buffer solution.

[0037] In some embodiments, at least one chelator is a divalent cation chelator, hi some embodiments, the divalent cation chelator is selected from the group consisting of EDTA, EGTA, CDTA, and citrate.

[0038] In some embodiments, the pH is increased to at least 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the pH is increased to at least about 7.2 to about 7.8. In some embodiments, the pH is increased to at least about 7.6. In some embodiments, the pH is increased by the addition of a basic amino acid. In some embodiments, the one or more wash buffers in b) comprise the one or more chelating agents. In some embodiments, the one or more wash buffers in b) exhibit a pH of at least 7. In some embodiments, the one or more wash buffers in b) comprise the one or more chelating agents and exhibit a pH of at least 7.

[0039] In some embodiments, the method further comprises a viral inactivation step, which is performed before, after, or simultaneously with the washing and / or elution steps, but before the recovery step. In some embodiments, the viral inactivation treatment inactivates lipid-enveloped viruses. In some embodiments, the viral inactivation treatment is a solvent and detergent (S / D) treatment.

[0040] In some embodiments, the one or more buffers comprise a buffer selected from the group consisting of glycine HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TrisHCl (tris(hydroxymethyl)-aminomethane), histidine, imidazole, acetate citrate, MES, and 2-(N-morpholino)ethanesulfonic acid.

[0041] In some embodiments, the one or more buffers further comprise one or more monovalent cations. In some embodiments, the one or more monovalent cations are selected from the group consisting of Na+, K+, Li+, and Cs+. In some embodiments, the monovalent cation is Na+.

[0042] In some embodiments, the one or more buffers further comprise one or more monovalent, divalent, and / or trivalent anions. In some embodiments, the one or more monovalent, divalent, and / or trivalent anions are Cl. - , acetic acid - , SO4 2- , Br - , and citric acid 3- is selected from the group consisting of:

[0043] In some embodiments, the one or more buffers comprise at least one buffer exhibiting a conductivity of ≧0.5 mS / cm at 25° C. In some embodiments, the one or more buffers comprise at least one buffer exhibiting a conductivity of 15.0±0.2 mS / cm at 25° C.

[0044] In some embodiments, the one or more buffers further comprise one or more non-ionic detergents, hi some embodiments, the non-ionic detergents are selected from the group consisting of Triton X-100, Tween 80, and Tween 20.

[0045] In some embodiments, the one or more buffers further comprise one or more additional substances selected from the group consisting of non-reducing sugars, sugar alcohols, and polyols.

[0046] In some embodiments, the highly pure mat-rVWF composition has a host cell (HC) impurity level of ≦2.0%. In some embodiments, the highly pure mat-rVWF composition has a host cell (HC) impurity level of ≦0.6%.

[0047] In some embodiments, the highly purified mat-rVWF composition is used in the manufacture of a pharmaceutical composition.

[0048] The present invention further provides a pharmaceutical composition comprising highly purified mat-rVWF produced by the method of any of the preceding claims and a pharmaceutically acceptable buffer. In some embodiments, the pharmaceutical composition comprises 50 mM glycine, 10 mM taurine, 5% (w / w) sucrose, 5% (w / w) D-mannitol, 0.1% polysorbate 80, 2 mM CaCl, 150 mM NaCl, and the pH of the composition is about pH 7.4.

[0049] Other objects, advantages and embodiments of the present invention will become apparent from the following detailed description. [Brief explanation of the drawings]

[0050] [Figure 1] 1 shows the purification of mature rVWF on a cation exchanger as described in Example 1. [Figure 2] A table of purification results is shown. [Figure 3] 1 shows a silver-stained protein gel and Western blot showing the separation of mat-VWF and r-VWF propeptide (rVWF-PP) by the method of Example 1. [Figure 4] 1 shows a flow chart of the experimental setup for Examples 2 and 3. [Figure 5] Chromatogram of Example 2 and chromatographic scheme used in Examples 2 and 3 are shown. [Figure 6] 1 shows a table of reagents used in Example 2 and a table of results. [Figure 7] 1 shows another chromatogram of Example 2 and a table of results of Example 3. [Figure 8] 1 shows a silver-stained protein gel showing the separation of mat-rVWF and rVWF propeptide (rVWF-PP) by the methods of Examples 2 and 3. [Figure 9] 1 shows a Western blot demonstrating the separation of rVWF and rVWF propeptide by the methods of Examples 2 and 3. [Figure 10] 1 shows the chromatogram, chromatography scheme, and buffer composition of Example 4. [Figure 11] 1 shows a table of results for Example 4. [Figure 12] 1 shows a silver-stained protein gel and Western blot showing the separation of mat-rVWF and rVWF propeptide (rVWF-PP) by the method of Example 4. [Figure 13] 1 shows the chromatogram, chromatography scheme, and buffer composition of Example 5. [Figure 14] 1 shows a table of results for Example 5. [Figure 15] 1 shows the chromatogram, chromatography scheme, and buffer composition of Example 6. [Figure 16] 1 shows a table of results for Example 6. [Figure 17] 1 shows the chromatogram, chromatography scheme, and buffer composition of Example 7. [Figure 18] 1 shows a table of results for Example 7. [Figure 19] 1 shows the chromatogram, chromatography scheme, and buffer composition of Example 8. [Figure 20] 1 shows a table of results for Example 8. [Figure 21] 1 shows a silver-stained protein gel showing the separation of mat-rVWF and rVWF propeptide (rVWF-PP) by the method of Example 8. [Figure 22] Figure 1 shows a Western blot demonstrating the separation of rVWF and rVWF propeptide by the method of Example 8. The 1% agarose gel shows the multimeric pattern of the products. [Figure 23] 1 shows a Western blot demonstrating the separation of mat-rVWF and rVWF propeptide (rVWF-PP) by the method of Example 8. [Figure 24] 1 shows the chromatogram, chromatography scheme, and buffer composition of Example 9. [Figure 25] 1 shows a table of results for Example 9. [Figure 26] 1 shows a table of products for Example 9. [Figure 27]10 shows a silver-stained protein gel showing the separation of rVWF and rVWF propeptide by the method of Example 9. [Figure 28] Figure 1 shows a Western blot demonstrating the separation of rVWF and rVWF propeptide by the method of Example 9. The 1% agarose gel shows the multimeric pattern of the products. [Figure 29] 1 shows a Western blot demonstrating the separation of rVWF and rVWF propeptide by the method of Example 9. [Figure 30] 1 shows the purity of the product-containing fractions obtained by enhanced cation exchange chromatography (CEX) used in Examples 1, 2, 3, 6, 8, and 9. [Figure 31] 1 shows the depletion factors of product-related impurities for Examples 1, 2, 3, 6, 8, and 9. [Figure 32] 1 shows the purity of the product-containing fractions obtained by enhanced size exclusion chromatography (SEC) used in Examples 4 and 5. [Figure 33] 1 shows the depletion factors of product-related impurities for Examples 4 and 5. [Figure 34] The buffer formulations and materials used in the TMAE separation method are shown. [Figure 35] The loading conditions for furin-treated mature VWF / VWF propeptide complexes are shown. [Figure 36] Details of the buffers, conditions, parameters, and flow rates of the chromatographic method are given. [Figure 37] Chromatogram of the dissociation of furin-treated mature VWF / VWF propeptide complex into mature VWF and VWF-propeptide (VWF-PP), showing depletion of VWF-PP from fractions containing mature VWF. [Figure 38] Another chromatogram of the separation of mature VWF and VWF-propeptide (VWF-PP) is shown, demonstrating the depletion of VWF-PP from fractions containing mature VWF. [Figure 39] Figures 39A and 39B show a schematic diagram of an exemplary method for purifying mature VWF, including separation of mature VWF and VWF-PP. [Figure 40]A table highlighting some of the advantages of the cation exchange chromatography method described herein is presented. [Figure 41] Figure 1 shows two schematic chromatograms illustrating the separation of rVWF propeptide using size exclusion chromatography as described herein using either SQA or SQC running buffers containing citric acid. Varying the SEC parameters (SEC buffer) resulted in increased removal / separation of residual VWF-PP while leaving mature VWF unpurified. [Figure 42] A table highlighting some of the advantages of the optimized SEC buffer (SQC buffer), which contains at least one chelating agent, has been shown to reduce the amount of VWF-PP in purified mature VWF fractions. [Figure 43A] Figure 43A shows a flow chart of the downstream processing protocol for rVWF. Figure 43A shows the process currently used. [Figure 43B] Figure 43B shows a flow chart of the downstream processing protocol for rVWF. Figure 43B shows the process described herein, including the improved CAT (UNO_S) step. [Figure 44] 1 shows a table of chromatography hardware for step CAT in the first generation (Gen1) and second generation (Gen2) processes. [Figure 45] A table of wash and elution conditions for the Gen2 process is shown. [Figure 46] A comparison table of the first and second generation rVWF small-scale polishing processes for UNO_Sphere S (process CAT) is shown below. [Figure 47] A table showing the cleaning and disinfection procedures for the UNO_Sphere S column is provided. [Figure 48] 1 shows a table of buffer compositions for the CAT polishing process. [Figure 49]Figures 49A and 49B show the chromatograms for run VW_USS_05. Figure 49A shows the entire chromatogram including the CIP procedure. Figure 49B shows the 36% Buffer B wash and gradient elution phase. UV absorption is shown in blue (280 nm) and magenta (254 nm). [Figure 50] Shown is an SDS-PAGE silver stained gel and Western blot of run VW_UUS_05. [Figure 51] Multimer agarose gel of run VW_UUS_05 is shown. [Figure 52] rVWF:Ag data from different runs of this study are shown. [Figure 53] rVWF Risto Co activity data from different runs of this study are shown. [Figure 54] Propeptide concentration (propeptide (μg / mg rVWF:Ag)) data for the different runs of this study are shown. [Figure 55] Data are shown for propeptide concentration (μg PP / 1000U Risto) for the different runs of this study. [Figure 56] The main analytical results of the eluate pools of the different runs of this study are shown. [Figure 57] The CAT-E criteria are presented as targets for successful method development. [Figure 58] 1 shows exemplary embodiments of anion exchange, cation exchange, and size exclusion chromatography methods for the separation of mat-rVWF and rVWF-PP. [Figure 59] The various VWF forms are shown: proVWF (also called pro-rVWF), matVWF / VWF-PP complex (also called mat-rVWF / VWF-PP complex), matVWF (also called mat-rVWF), and VWF-PP (also called rVWF-PP). [Figure 60-1] The nucleic acid and amino acid sequences of VWF are shown. [Figure 60-2] See description of Figure 60-1. [Figure 60-3] See description of Figure 60-1. [Figure 60-4] See description of Figure 60-1. [Figure 60-5] See description of Figure 60-1. [Figure 60-6] See description of Figure 60-1. [Figure 60-7] See description of Figure 60-1. [Figure 60-8] See description of Figure 60-1. [Figure 60-9] See description of Figure 60-1. [Figure 60-10] See description of Figure 60-1. [Figure 60-11] See description of Figure 60-1. [Figure 60-12] See description of Figure 60-1. [Figure 60-13] See description of Figure 60-1. [Figure 60-14] See description of Figure 60-1. [Figure 60-15] See description of Figure 60-1. [Figure 60-16] See description of Figure 60-1. [Figure 60-17] See description of Figure 60-1. [Figure 60-18] See description of Figure 60-1. [Figure 61] Analytical DF3338 / 042 Western blot and raw data are shown. [Figure 62] Analytical DF3362 / 023 Western blot and raw data are shown. [Figure 63] A comparison of the data in Figures 61 and 62 is shown. [Figure 64-1] The amino acid sequence of a representative VWF-FVIII fusion protein is shown, in which active FVIII is embedded in a VWF motif (VWF764-1336-FVIII heavy chain 24-760-VWF2218-2593-FVIII light chain 1333-2351-VWF2620-2813). [Figure 64-2] See description of Figure 64-1. [Figure 64-3] See description of Figure 64-1. [Figure 65-1] 1 shows the amino acid sequence of an exemplary VWF-FVIII fusion protein, in which the FVIII-B domain is replaced with an n-glycosylation-rich domain (FVIII heavy chain 19-760-vWF2218-2593-FVIII light chain 1333-2351). [Figure 65-2] See description of Figure 65-1. [Figure 65-3] See description of Figure 65-1. [Figure 66] 1 shows a table of buffers and compositions used in the purification process of variant vWF described in Example 14. [Figure 67] The chromatogram and chromatogram scheme for run VW_USS_07 are shown. [Figure 68] The main analytical results of the implementation are presented. [Figure 69] An SDS-PAGE silver stained gel of a representative run is shown. Depletion of rvWF-propeptide was observed during the washing steps Wash1, WSD, and Wash2. [Figure 70] Figure 1 shows a table of buffers and compositions used in the purification process of variant vWF described in Example 15. This example provides an alternative variant embodiment for separating the r-vWF propeptide from the r-VWF polypeptide after furin cleavage to test further sialylation. [Figure 71] Chromatogram and chromatogram scheme for run VW_USS_06 are shown. [Figure 72] The main analytical results of the implementation are presented. [Figure 73] An SDS-PAGE silver stained gel of a representative run is shown. [Figure 74] 1 shows a table of buffers and compositions used in the purification process of variant vWF described in Example 16. [Figure 75] Chromatogram and chromatogram scheme for run VW_USS_08 are shown. [Figure 76] Key analytical results of the run are shown, including sialylation yield. [Figure 77]An SDS-PAGE silver stained gel of a representative run DFM07247 is shown. [Figure 78] Figure 1 shows the sialylation profile of the eluates from runs VW_USS_06 and VW_USS_08. DETAILED DESCRIPTION OF THE INVENTION

[0051] Detailed Description of the Invention I. Introduction The methods described herein separate dissociated mature VWF and VWF propeptide from non-covalently bound heterodimeric complexes containing mature VWF and VWF propeptide. This separation is facilitated (induced) by adding at least one chelating agent to the protein separation method and / or by increasing the pH of the solution containing mature VWF and VWF propeptide to at least 7.0. All of the enhanced anion exchange (AEX), cation exchange (CEX), and / or size exclusion chromatography (SEC) methods described herein, in any variation, can be combined to obtain r-vWF with improved properties.

[0052] II. Choice of definition The term "recombinant," when applied to, for example, a cell, or a nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, a recombinant cell expresses genes that are not found in the native (non-recombinant) form of the cell, or expresses native genes in aberrant, reduced, or absent ways.

[0053] As used herein, "recombinant VWF" or "rVWF" includes VWF obtained via recombinant DNA technology. In certain embodiments, the rVWF protein of the present invention may comprise a construct, for example, as described in U.S. Pat. No. 8,597,910, which is incorporated herein by reference, for methods of producing recombinant VWF. VWF in the present invention may include all potential forms, including monomeric and multimeric forms. It should also be understood that the present invention encompasses different forms of VWF used in combination. For example, the VWF of the present invention may include different multimers, different derivatives, and both biologically active and non-biologically active derivatives.

[0054] In the context of the present invention, recombinant VWF includes any member of the VWF family from mammals, such as primates, humans, monkeys, rabbits, pigs, rodents, mice, rats, hamsters, gerbils, dogs, and cats, as well as biologically active derivatives thereof. Active mutant and variant VWF proteins are also encompassed, as are functional fragments and fusion proteins of VWF proteins. Furthermore, the VWF of the present invention may further comprise tags that facilitate purification, detection, or both. The VWF described herein may also be modified with moieties suitable for therapeutic or imaging purposes in vitro or in vivo.

[0055] The term "VWF multimer" refers to a VWF containing at least 10 subunits, or 12, 14, or 16 subunits to about 20, 22, 24, or 26 or more subunits. The term "subunit" refers to a VWF multimer. As known in the art, it is generally a VWF dimer that polymerizes to form higher-order multimers. (See, e.g., Turecek et al., Semin. Thromb. Hemost., 2010, 36(5):510-521, which is incorporated herein by reference in its entirety for all purposes, particularly for all teachings regarding VWF multimer analysis.)

[0056] The terms "prepropeptide VWF," "preproVWF," or "proVWF" refer to a non-mature VWF polypeptide containing a signal peptide of approximately 22 amino acid residues, a VWF propeptide of approximately 741 amino acid residues, and a mature VWF subunit of approximately 2050 amino acid residues. The proVWF subunit can dimerize through disulfide bonds near the carboxyl terminus of the endoplasmic reticulum to form tail-to-tail dimers, which are then transported to the Golgi apparatus. In the Golgi apparatus, an additional head-to-head disulfide bond is formed proximal to the amino terminus of the subunit, resulting in the formation of multimers. Proteolytic cleavage of the VWF propeptide occurs via the processing protease furin, thus generating the mature VWF / VWF-PP complex. The inclusion of an "r" before the name of a VWF indicates that it is a recombinant version. In some embodiments, the methods described herein are applied to recombinant VWF (rVWF).

[0057] The term "VWF complex" or "mat-VWF / VWF-PP complex" refers to a non-covalent heterodimeric structure comprising a mature VWF subunit and a VWF propeptide. The VWF complex may be generated as a product of furin cleavage between the propeptide portion of prepropeptide VWF and the mature VWF portion. When the name of VWF includes an "r" before it, it indicates that it is a recombinant version. In some embodiments, the methods described herein are applied to recombinant VWF (rVWF).

[0058] The term "mature VWF" or "mat-VWF" refers to a mature VWF subunit of approximately 2050 amino acid residues. The mature VWF subunit may be part of a prepropeptide VWF or a VWF complex. Mature VWF may be referred to as "free VWF" upon separation (isolation) from the VWF propeptide. When the name of a VWF includes an "r" before it, it indicates that it is a recombinant version. In some embodiments, the methods described herein are applied to recombinant VWF (rVWF).

[0059] The term "VWF propeptide" or "VWF-PP" refers to a VWF propeptide of approximately 741 amino acid residues. The VWF propeptide may be part of a prepropeptide VWF or a VWF complex. For example, in a VWF complex, the VWF propeptide is non-covalently associated with a mature VWF subunit. The VWF propeptide may be referred to as a "free VWF propeptide" when separated (isolated) from mature VWF. When the name of a VWF includes an "r" before it, it indicates that it is a recombinant version. In some embodiments, the methods described herein are applied to recombinant VWF (rVWF).

[0060] The terms "isolated," "purified," or "biologically pure" refer to material that is substantially or essentially free from components that normally accompany it in its native state. Purity and homogeneity are typically measured using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. VWF is the predominant species present in a substantially purified preparation. In some embodiments, the term "purified" indicates that the nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. In other embodiments, the term refers to a nucleic acid or protein that is at least 50% pure, more preferably at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more pure. In other embodiments, "purifying" or "purifying" refers to the removal of at least one contaminant from the composition being purified. In this sense, purification does not require that the purified compound be homogeneous, e.g., 100% pure.

[0061] As used herein, the term "about" indicates an approximate range of ±10% from the specified value. For example, the phrase "about 20%" encompasses a range of 18-22%.

[0062] III. Detailed Description of the Embodiments The present invention relates to a method for obtaining a highly pure composition containing free mature recombinant von Willebrand factor (rVWF), the method comprising the following steps: dissociating mature rVWF from rVWF propeptide using a solution (e.g., a dissociation solution) containing at least one chelating agent or having a pH of at least 7; separating free mature rVWF from rVWF propeptide; and recovering a free mature rVWF composition containing at least 95% free mature rVWF and less than 5% rVWF propeptide.

[0063] The methods of the present invention are particularly suited for the in vitro separation of mature VWF from its VWF propeptide. In some embodiments, separation is induced by adding one or more chelating agents to a solution containing mature VWF and VWF-PP, by raising the pH of the solution to at least 7.0, or a combination thereof. In some embodiments, the pH is raised to a range of pH 7.0 to pH 9.0.

[0064] The separation method can include, but is not limited to, the use of one or more protein separation methods, such as, for example, chromatography methods, to isolate mature VWF from VWF-PP. This method can produce a highly pure free mature rVWF composition. In some embodiments, the free mature rVWF composition contains at least 95% free mature rVWF and less than 5% free rVWF-PP and / or matVWF / VWF-PP complexes. In some cases, the free mature rVWF composition comprises at least 96% free mature rVWF and less than 4% free rVWF-PP and / or matVWF / VWF-PP complexes, at least 97% free mature rVWF and less than 3% free rVWF-PP and / or matVWF / VWF-PP complexes, at least 98% free mature rVWF and less than 2% free rVWF-PP and / or matVWF / VWF-PP complexes, at least 99% free mature rVWF-PP and less than 1% free rVWF-PP and / or matVWF / VWF-PP complexes, at least 99.5% free mature rVWF and less than 0.5% free rVWF-PP / PP and / or matVWF / VWF-PP complexes.

[0065] a. Anion exchange chromatography purification In one embodiment of this method, mature rVWF (mat-rVWF) is separated from rVWF-PP using anion exchange (AEX) chromatography. In some cases, residual host cell-derived impurities such as CHO host cell proteins, process-related impurities such as recombinant furin and low molecular weight virus inactivation reagents, media compounds such as soy peptone, and other product-related impurities are removed from the mature VWF.

[0066] In another aspect of the method, anion exchange chromatography is used to separate mature rVWF from rVWF-PP, e.g., residual rVWF-PP or free rVWF-PP. For separation, the starting composition, loading solution, or loading composition can contain a low pH and at least one chelating agent. The loading composition can be added to an anion exchanger operating in flow-through mode. In some embodiments, the loading solution contains pro-rVWF, mat-rVWF / rVWF-PP complex, mat-rVWF, and / or rVWF propeptide (rVWF-PP). In some embodiments, the anion exchanger is operated in binding mode, and mature VWF and VWF-PP are separated using a gradient elution buffer containing at least one chelating agent. In other embodiments, the gradient elution buffer has a pH ranging from neutral to high, e.g., pH 6.0 to pH 9.0. In another embodiment, the gradient elution buffer contains one or more chelating agents and has a pH of 7.0 or higher, for example, a pH of 7.0 to 9.0. For example, the gradient elution buffer may contain EDTA and have a pH of 8.5.

[0067] In some embodiments, the present invention provides a method for obtaining a composition comprising highly purified mature recombinant rVWF depleted of propeptide (highly purified mat-rVWF), the method comprising the following steps: (a) loading a solution containing pro-rVWF, mat-rVWF / rVWF-PP complex, mat-rVWF, and / or rVWF propeptide (rVWF-PP) onto an anion exchange column to bind the pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF to the anion exchange column; (b) washing the anion exchange column of (a) containing the bound pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF with one or more wash buffers; and (c) treating the column of (b) containing the bound pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF with furin. (d) eluting the bound pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF from the column of (c) with an elution buffer, wherein the elution buffer induces dissociation of the rVWF-PP from mat-rVWF non-covalently associated with the rVWF-PP, and the dissociation is induced by (i) adding at least one chelating agent to the elution buffer or (ii) increasing the pH of the elution buffer to at least pH 7; and (e) separating and recovering the mat-rVWF from the rVWF-PP to obtain a highly purified mat-rVWF composition, wherein the highly purified mat-rVWF composition comprises at least 95% mature rVWF and less than 5% rVWF-PP.

[0068] In some embodiments, a) and b) are performed simultaneously in a single step. In some embodiments, the solution in a) comprises flow-through from an immunoaffinity purification method. In some embodiments, the solution in a) comprises flow-through from a monoclonal antibody column, wherein the monoclonal antibody is a FVIII monoclonal antibody. In some embodiments, the solution in a) is selected from the group consisting of cell culture medium, an antibody column flow-through solution, and a buffer solution.

[0069] In some embodiments of step (b), washing the anion exchange column of a) containing the bound pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF uses washing with one or more wash buffers, where the one or more wash buffers include one, two, three, four, and / or five wash buffers. In some embodiments, the second wash buffer contains components for virus inactivation. In some embodiments, when four or five wash buffers are used, the second wash buffer contains components for virus inactivation. In some embodiments, when four or five wash buffers are used, the second or third wash buffer contains components for a virus inactivation treatment. In some embodiments, the virus inactivation treatment is a solvent and detergent (S / D) treatment. In some embodiments, when five wash buffers are used, the first, second, third, and / or fifth wash buffer have a higher pH than the fourth wash buffer. In some embodiments, when five wash buffers are used, the first, second, third, and fifth wash buffers have a pH of about pH 7 to pH 8, and the fourth wash buffer has a pH of about pH 5 to pH 6. In some embodiments, when five wash buffers are used, the first, second, third, and / or fifth wash buffers have a pH of about pH 7.4 to pH 7.5, and the fourth wash buffer has a pH of about pH 5.5. In some embodiments, the virus inactivation step is performed in a buffer having a higher pH than the fourth wash buffer. In some embodiments, when four wash buffers are used, the virus inactivation step is performed after the first wash buffer. In some embodiments, when four wash buffers are used, the first, second, and fourth wash buffers have a higher pH than the third wash buffer. In some embodiments, the virus inactivation step is performed in a buffer having a higher pH than the third wash buffer. In some embodiments, the virus inactivation step is performed in a buffer having the same pH as the first, second, and / or fourth wash buffers. In some embodiments, when four wash buffers are used, the first, second, and fourth wash buffers have a pH of about pH 7 to about pH 8, and the third wash buffer has a pH of about pH 5 to about pH 6.In some embodiments, when four wash buffers are used, the first, second, and fourth wash buffers have a pH of about pH 7.4 to pH 7.5, and the third wash buffer has a pH of about pH 5.5.

[0070] Anion exchange chromatography can be performed as recognized by those skilled in the art. In some embodiments, the anion exchanger includes, but is not limited to, STREAMLINE Q XL™, POROS 50 PI™, Q SEPHAROSE™, Emphase™ AEX Hybrid Purifier, Nuvia Q, POROS 50 HQ, Capto Q, Capto Q impress, Unosphere Q, Q Ceramic HYPERD™ F, TOYOPEARL™ Q, TOYOPEARL™ Super Q, mixed-mode AEX resins (e.g., Capto Adhere, Capto adhere impress, or MEP Hypercell), and any DEAE, TMAE, tertiary or quaternary amine, or PEI-based resin. In some embodiments, the anion exchanger is a membrane anion exchanger. In some embodiments, the membrane anion exchanger includes, but is not limited to, Sartobind Q®, Sartobind STIC® PA, Mustang Q®, or ChromaSorb®. In some embodiments, the anion exchanger is a Fractogel TMAE column (Merck-Millipore) or its equivalent.

[0071] In some embodiments, the loading concentration of pro-VWF is about 90 IU / ml to about 270 IU / ml resin, e.g., about 90 IU / ml to about 270 IU / ml, about 100 IU / ml to about 270 IU / ml, about 110 IU / ml to about 270 IU / ml, about 120 IU / ml to about 270 IU / ml, about 13 ... ml ~ approx. 270IU / ml, approx. 140IU / ml ~ approx. 270IU / ml, approx. 150IU / ml ~ approx. 270IU / ml, approx. 90IU / ml ~ approx. 250IU / ml, approx. 100IU / ml ml ~ approx. 250IU / ml, approx. 110IU / ml ~ approx. 250IU / ml, approx. 120IU / ml ~ approx. 250IU / ml, approx. 130IU / ml ~ approx. 250IU / ml, approx. 130IU / ml ml ~ approx. 250IU / ml, approx. 140IU / ml ~ approx. 250IU / ml, approx. 150IU / ml ~ approx. 250IU / ml, approx. 90IU / ml ~ approx. 200IU / ml, approx. 100IU / ml ml ~ approx. 200IU / ml, approx. 110IU / ml ~ approx. 200IU / ml, approx. 120IU / ml ~ approx. 200IU / ml, approx. 130IU / ml ~ approx. 200IU / ml, approx. 130IU / ml ml to about 200 IU / ml, about 140 IU / ml to about 200 IU / ml, about 150 IU / ml to about 200 IU / ml, about 90 IU / ml to about 100 IU / ml, about 100 IU / ml to about 150 IU / ml, about 150 IU / ml to about 200 IU / ml, about 200 IU / ml to about 250 IU / ml, or about 250 IU / ml to about 270 IU / ml resin.

[0072] In some embodiments, the anion exchange method comprises a buffer system. In some embodiments, the buffer system comprises one or more elution buffers. In some embodiments, the buffer system comprises one or more wash buffers. In some embodiments, the buffer system comprises at least one elution buffer and at least one wash buffer. In some embodiments, the buffer system comprises at least two elution buffers and at least two wash buffers.

[0073] In some embodiments, the loading concentration is from about 90 IU / ml to about 270 IU / ml resin, e.g., from about 90 IU / ml to about 270 IU / ml, from about 100 IU / ml to about 270 IU / ml, from about 110 IU / ml to about 270 IU / ml, from about 120 IU / ml to about 270 IU / ml, from about 130 IU / ml to about 270 IU / ml, from about 130 IU / ml to about 270IU / ml, about 140IU / ml to about 270IU / ml, about 150IU / ml to about 270IU / ml, about 90IU / ml to about 250IU / ml, about 100IU / ml to about 250IU / ml, about 110IU / ml~about 250IU / ml, about 120IU / ml~about 250IU / ml, about 130IU / ml~about 250IU / ml, about 130IU / ml~ Approx. 250IU / ml, approx. 140IU / ml ~ approx. 250IU / ml, approx. 150IU / ml ~ approx. 250IU / ml, approx. 90IU / ml ~ approx. 200IU / ml, approx. 100IU / ml ~ Approx. 200IU / ml, approx. 110IU / ml ~ approx. 200IU / ml, approx. 120IU / ml ~ approx. 200IU / ml, approx. 130IU / ml ~ approx. 200IU / ml, approx. 130IU / ml about 140 IU / ml to about 200 IU / ml, about 150 IU / ml to about 200 IU / ml, about 90 IU / ml to about 100 IU / ml, about 100 IU / ml to about 150 IU / ml, about 150 IU / ml to about 200 IU / ml, about 200 IU / ml to about 250 IU / ml, or about 250 IU / ml to about 270 IU / ml resin.

[0074] In some embodiments, the pH of the starting composition, loading solution, or loading composition comprising pro-rVWF, mat-rVWF / rVWF-PP complex, mat-rVWF, and / or rVWF propeptide (rVWF-PP) is between pH 6.0 and pH 9.0, e.g., pH 6.0 and pH 9.0, pH 6.3 and pH 9.0, pH 6.5 and pH 9.0, pH 7.0 and pH 9.0, pH 7.5 and pH 9.0, pH 7.7 and pH 9.0, pH 8.0 and pH 9.0, pH 6.0 and pH 8.5, pH 6.5 and pH 8.5, pH 7.0 ... .5 to pH8.5, pH6.0 to pH8.0, pH6.5 to pH8.0, pH7.0 to pH8.0, pH7.5 to pH8.0, pH6.0, pH6.1, pH6.2, pH6.3, pH6.4, pH6.5, pH6.6, pH6.7, pH6.8, pH6.9, pH7.0, pH7.1, pH7.2, pH7.3, pH7.4, pH7.5, pH7.6, pH7.7, pH7.8, pH7.9, pH8.0, pH8.1, pH8.2, pH8.3, pH8.4, pH8.5, pH8.6, pH8.7, pH8.8, pH8.9, or pH9.0.

[0075] In some embodiments, the conductivity of the starting composition, loading solution, or loading composition comprising pro-rVWF, mat-rVWF / rVWF-PP complex, mat-rVWF, and / or rVWF propeptide (rVWF-PP) is between about 5 mS / cm and about 40 mS / cm, e.g., between about 5 mS / cm and about 40 mS / cm, between about 10 mS / cm and about 40 mS / cm, between about 15 mS / cm to about 40 mS / cm, about 20 mS / cm to about 40 mS / cm, about 25 mS / cm to about 40 mS / cm, about 30 mS / cm to about 40 mS / cm, about 10 mS / cm to about 40 mS / cm, about 10 mS / cm to about 30 mS / cm, about 5 mS / cm to about 15 mS / cm, about 15 mS / cm to about 30 mS / cm, or about 20 mS / cm to about 40 mS / cm.

[0076] In some embodiments, a starting composition, loading solution, or loading composition comprising pro-rVWF, mat-rVWF / rVWF-PP complex, mat-rVWF, and / or rVWF propeptide (rVWF-PP) is added to a solution of sodium citrate, such as, but not limited to, 10 mM to 80 mM sodium citrate, 15 mM to 80 mM sodium citrate, 10 mM to 80 mM sodium citrate, 15 mM to 60 mM citrate, Dilute with buffer containing sodium citrate, 20 mM - 60 mM sodium citrate, 10 mM sodium citrate, 20 mM sodium citrate, 30 mM sodium citrate, 40 mM sodium citrate, 50 mM sodium citrate, 55 mM sodium citrate, 60 mM sodium citrate, 65 mM sodium citrate, 70 mM sodium citrate, 75 mM sodium citrate, 80 mM sodium citrate, etc.

[0077] In some embodiments, the first wash buffer comprises at least one chelating agent and optionally has a pH in the range of pH 6.0 to pH 9.0. In some embodiments, the first wash buffer comprises a pH in the range of pH 6.0 to pH 9.0 and optionally comprises at least one chelating agent. In some embodiments, the first wash buffer comprises a pH in the range of pH 6.0 to pH 6.9. In some embodiments, the second wash buffer comprises a pH in the range of pH 7.0 to pH 9.0. In some embodiments, the first wash buffer can comprise at least one chelating agent and has a pH in the range of pH 6.0 to pH 6.9. In some embodiments, the wash elution buffer has a pH less than 7. In one embodiment, the second wash buffer has a pH greater than 7. In some embodiments, when two wash buffers are used, the first wash buffer has a pH less than 7 and the second wash buffer has a pH greater than 7.

[0078] In some embodiments, one or more wash buffers are selected from the group consisting of 120 mM to 200 mM, 130 mM to 200 mM, 140 mM to 200 mM, 150 mM to 200 mM, 120 mM to 190 mM, 130 mM to 190 mM, 140 mM to 190 mM, 150 mM to 190 mM, 120 mM to 180 mM, 130 mM to 180 mM, with NaCl concentrations of 100 mM, 140 mM to 180 mM, 150 mM to 180 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, or 200 mM.

[0079] In some embodiments, an initiating composition, loading solution, or loading composition comprising mature VWF and VWF-PP is contacted with a buffer comprising at least one chelating agent, optionally having a pH in the range of pH 6.0 to pH 9.0. In some embodiments, an initiating composition, loading solution, or loading composition is contacted with a buffer comprising at least one chelating agent, optionally having a pH in the range of pH 6.0 to pH 9.0. In some embodiments, an initiating composition, loading solution, or loading composition is contacted with a buffer comprising at least one chelating agent, optionally having a pH in the range of pH 6.0 to pH 9.0. In some embodiments, the buffer is a wash buffer. In some embodiments, the buffer is an elution buffer. In some embodiments, the buffer is a wash buffer having a pH of 6.0 to 6.9. In some embodiments, the buffer is an elution buffer having a pH of 7.0 to 9.0. In some embodiments, an initiating composition, loading solution, or loading composition comprising mature VWF and VWF-PP is contacted first with a wash buffer having a pH of 6.0 to 6.9 and second with at least one elution buffer having a pH of 7.0 to 9.0.

[0080] In some embodiments, mature VWF is eluted in the anion exchange chromatography step using a single elution buffer. In some embodiments, mature VWF is eluted in the anion exchange chromatography step using a gradient elution method comprising multiple elution buffers. For example, elution can be performed using two elution buffers, such as a first elution buffer and a second elution buffer. In some embodiments, the first elution buffer comprises at least one chelating agent and optionally has a pH in the range of pH 6.0 to pH 9.0. In some embodiments, the first elution buffer has a pH in the range of pH 6.0 to pH 9.0 and optionally comprises at least one chelating agent. In some embodiments, the first elution buffer has a pH in the range of pH 6.0 to pH 6.9. In some embodiments, the second elution buffer has a pH in the range of pH 7.0 to pH 9.0. In some embodiments, the first elution buffer can comprise at least one chelating agent and has a pH in the range of pH 6.0 to pH 6.9. In some embodiments, the first elution buffer has a pH less than 7. In one embodiment, the second elution buffer has a pH greater than 7. In some embodiments, when two elution buffers are used, the first elution buffer has a pH less than 7 and the second elution buffer has a pH greater than 7.

[0081] In some embodiments, the pH of the wash buffer for the anion exchange chromatography step is between pH 6.0 and pH 9.0, e.g., pH 6.0 and pH 9.0, pH 6.3 and pH 9.0, pH 6.5 and pH 9.0, pH 7.0 and pH 9.0, pH 7.5 and pH 9.0, pH 7.7.0 and pH 9.0, pH 8.0 and pH 9.0, pH 6.0 and pH 8.5, pH 6.5 and pH 8.5, pH 7.0 and pH 8.5, pH 7.5 and pH 8.5, pH 6.0 and pH 8.0, pH 6.5 and pH 8.0 , pH 7.0 to pH 8.0, pH 7.5 to pH 8.0, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, pH 9.0. In some embodiments, this includes when two elution buffers are present, e.g., first and second elution buffers.

[0082] In some embodiments, the pH of the elution buffer for the anion exchange chromatography step is between pH 6.0 and pH 9.0, e.g., pH 6.0 and pH 9.0, pH 6.3 and pH 9.0, pH 6.5 and pH 9.0, pH 7.0 and pH 9.0, pH 7.5 and pH 9.0, pH 7.7.0 and pH 9.0, pH 8.0 and pH 9.0, pH 6.0 and pH 8.5, pH 6.5 and pH 8.5, pH 7.0 and pH 8.5, pH 7.5 and pH 8.5, pH 6.0 and pH 8.0, pH 6.5 and pH 8.0 , pH 7.0 to pH 8.0, pH 7.5 to pH 8.0, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, pH 9.0. In some embodiments, this includes when two elution buffers are present, e.g., first and second elution buffers.

[0083] In some embodiments, the pH of the elution buffer is increased relative to the starting solution of step a), increased relative to the first elution buffer if two elution buffers are used, and / or increased relative to the wash buffer if a wash buffer is used. In some embodiments, when a wash buffer and an elution buffer are used, the wash buffer has a pH less than 7 and the elution buffer has a pH greater than 7. In some embodiments, when two elution buffers are used, one elution buffer has a pH less than 7 and the other elution buffer has a pH greater than 7. In some embodiments, when a wash buffer and two elution buffers are used, the wash buffer has a pH less than 7 and both elution buffers have a pH greater than 7. In some embodiments, when a wash buffer and two elution buffers are used, the wash buffer and the first elution buffer have a pH less than 7 and the second elution buffer have a pH greater than 7. In some embodiments, when two wash buffers and two elution buffers are used, the wash buffer and the first elution buffer have a pH less than 7 and the second elution buffer have a pH greater than 7. In some embodiments, when two wash buffers and two elution buffers are used, both wash buffers have a pH less than 7 and both elution buffers have a pH greater than 7. In some embodiments, when two wash buffers and two elution buffers are used, the first wash buffer has a pH below 7, and the second wash buffer and both elution buffers have a pH above 7.

[0084] In some embodiments, as described in step (a) of the method, the pH of one or more wash buffers and / or elution buffers is increased to at least 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0 compared to the loading solution containing pro-rVWF, mat-rVWF / rVWF-PP complex, mat-rVWF, and / or rVWF propeptide (rVWF-PP). In some embodiments, to induce dissociation of the mat-rVWF / rVWF-PP complex into mat-rVWF and rVWF-PP in the solution of step (a) of the method, the pH of the buffer is increased to at least 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, where the dissociation occurs by disruption of non-covalently associated mat-rVWF and rVWF-PP. In some embodiments, the pH of the loading solution is increased to at least about 7.2 to about 7.8. In some embodiments, the pH of the loading solution is increased to at least about 7.6. In some embodiments, the pH of the loading solution is increased by the addition of a basic amino acid. In some embodiments, the pH of the loading solution is increased to at least 7. In some embodiments, the pH of one or more wash buffers is increased to at least about 7.2 to about 7.8. In some embodiments, the pH of one or more wash buffers is increased to at least about 7.6. In some embodiments, the pH of one or more wash buffers is increased by the addition of a basic amino acid. In some embodiments, one or more wash buffers exhibit a pH of at least 7. In some embodiments, the pH of one or more elution buffers is increased to at least about 7.2 to about 7.8. In some embodiments, the pH of one or more elution buffers is increased to at least about 7.6. In some embodiments, the pH of one or more elution buffers is increased by the addition of a basic amino acid. In some embodiments, one or more elution buffers exhibit a pH of at least 7.

[0085] In some embodiments, one or more buffers (including wash buffers and / or elution buffers) comprise one or more chelating agents. In some embodiments, the elution buffer comprises at least one chelating agent. The chelating agent can be a divalent cation chelating agent. In some embodiments, the at least one chelating agent is a divalent cation chelating agent. In some embodiments, the divalent cation chelating agent is selected from the group consisting of EDTA, EGTA, CDTA, and citrate. In some embodiments, the divalent cation chelating agent is selected from the group consisting of NTA, DTPA, EDDS, EDTA, EGTA, CDTA, and citrate. In some embodiments, the chelating agent is NTA. In some embodiments, the chelating agent is DTPA. In some embodiments, the chelating agent is EDDS. In some embodiments, the chelating agent is EDTA. In some embodiments, the chelating agent is EGTA. In some embodiments, the chelating agent is CDTA. In some embodiments, the chelating agent is citrate. In some embodiments, the one or more wash buffers in b) comprise the one or more chelating agents and exhibit a pH of at least 7.

[0086] In some embodiments, one or more buffers (including wash and / or elution buffers) comprise sodium citrate in a range including, but not limited to, 10 mM to 80 mM sodium citrate, 15 mM to 80 mM sodium citrate, 10 mM to 80 mM sodium citrate, 15 mM to 60 mM sodium citrate, 20 mM to 60 mM sodium citrate, 10 mM sodium citrate, 20 mM sodium citrate, 30 mM sodium citrate, 40 mM sodium citrate, 50 mM sodium citrate, 55 mM sodium citrate, 60 mM sodium citrate, 65 mM sodium citrate, 70 mM sodium citrate, 75 mM sodium citrate, 80 mM sodium citrate, etc.

[0087] In some embodiments, the first elution buffer further comprises sodium citrate in a range including, but not limited to, 10 mM to 60 mM sodium citrate, 15 mM to 60 mM sodium citrate, 10 mM to 50 mM sodium citrate, 15 mM to 50 mM sodium citrate, 20 mM to 60 mM sodium citrate, 10 mM sodium citrate, 20 mM sodium citrate, 30 mM sodium citrate, 40 mM sodium citrate, 50 mM sodium citrate, 60 mM sodium citrate, etc.

[0088] In some embodiments, the second elution buffer further comprises sodium citrate, for example, but not limited to, 10 mM to 60 mM sodium citrate, 15 mM to 60 mM sodium citrate, 10 mM to 50 mM sodium citrate, 15 mM to 50 mM sodium citrate, 20 mM to 60 mM sodium citrate, 10 mM sodium citrate, 20 mM sodium citrate, 30 mM sodium citrate, 40 mM sodium citrate, 50 mM sodium citrate, 60 mM sodium citrate, etc.

[0089] In some embodiments, elution buffer A and / or elution buffer B in the anion exchange chromatography step may be from about 0.5 mM to about 20 mM EDTA, for example, about 0.5mM to about 20mM, about 1mM to about 20mM, about 1.5mM to about 20mM, about 2mM to about 20mM, about 3m M ~ about 20mM, about 5mM - about 20mM, about 0.5mM - about 15mM, about 1mM - about 10mM, about 1mM - about 5mM, about 5mM, about 0. 5mmm, about 1mM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11m M, including about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM, etc.

[0090] In some embodiments, citrate can be found in the eluate after removal of the rVWF propeptide using an anion exchange method. In some embodiments, citrate can be found in the eluate after removal of the rVWF propeptide using a stepwise anion exchange elution method. In some embodiments, citrate can be found in the eluate after removal of the rVWF propeptide using a gradient anion exchange elution method. In some embodiments, the anion exchange counterion is citrate. 3- is.

[0091] Any of the buffers (buffer systems) described herein can be selected from the group consisting of glycine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TrisHCl (tris(hydroxymethyl)-aminomethane), histidine, imidazole, acetate citrate, citrate, acetate, MES, phosphate, TrisHCl, Bis-Tris, histidine, imidazole, arginineHCl, lysineHCl, and 2-(N-morpholino)ethanesulfonic acid, either as a single buffer or as a combination of two or more buffers. In some embodiments, the buffer comprises glycine. In some embodiments, the buffer comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). In some embodiments, the buffer comprises TrisHCl (tris(hydroxymethyl)-aminomethane). In some embodiments, the buffer comprises histidine. In some embodiments, the buffer comprises imidazole. In some embodiments, the buffer comprises acetate citrate. In some embodiments, the buffer comprises citrate. In some embodiments, the buffer comprises acetate. In some embodiments, the buffer comprises MES. In some embodiments, the buffer comprises phosphate. In some embodiments, the buffer comprises Tris-HCl. In some embodiments, the buffer comprises Bis-Tris. In some embodiments, the buffer comprises histidine. In some embodiments, the buffer comprises imidazole. In some embodiments, the buffer comprises arginine-HCl. In some embodiments, the buffer comprises lysine-HCl. In some embodiments, the buffer comprises 2-(N-morpholino)ethanesulfonic acid. In some embodiments, the buffer comprises one, two, three, or four of the buffers described herein.

[0092] In some embodiments, the one or more buffers are selected from the group consisting of glycine HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TrisHCl (tris(hydroxymethyl)-aminomethane), histidine, imidazole, acetate citrate, MES, and 2-(N-morpholino)ethanesulfonic acid.

[0093] In some embodiments, the one or more buffers comprise at least one buffer exhibiting a conductivity of ≧0.5 mS / cm at 25° C. In some embodiments, the one or more buffers comprise at least one buffer exhibiting a conductivity of 20.0±0.2 mS / cm at 25° C. In some embodiments, the one or more buffers comprise at least one buffer exhibiting a conductivity of 17.0±0.2 mS / cm at 25° C. In some embodiments, the one or more buffers comprise at least one buffer exhibiting a conductivity of 15.0±0.2 mS / cm at 25° C. In some embodiments, the one or more buffers comprise at least one buffer exhibiting a conductivity of 12.0±0.2 mS / cm at 25° C. In some embodiments, the one or more buffers comprise at least one buffer exhibiting a conductivity of 10.0±0.2 mS / cm at 25° C. In some embodiments, the one or more buffers comprise at least one buffer exhibiting a conductivity of 5.0±0.2 mS / cm at 25° C. In some embodiments, the one or more buffers comprise at least one buffer that exhibits a conductivity of 2.0±0.2 mS / cm at 25°C.

[0094] In some embodiments, the flow rate of one or more wash steps of the method is from about 10 cm / hr to about 200 cm / hr, e.g., about 10 cm / hr, about 15 cm / hr, about 20 cm / hr, about 25 cm / hr, about 30 cm / hr, about 35 cm / hr, about 40 cm / hr, about 45 cm / hr, about 50 cm / hr, about 55 cm / hr, about 60 cm / hr, about 65 cm / hr, about 70 cm / hr, about 75 cm / hr, about 80 cm / hr, about 85 cm / hr, about 90 cm / hr, about 95 cm / hr, The flow rate can be about 100 cm / hr, about 105 cm / hr, about 110 cm / hr, about 115 cm / hr, about 120 cm / hr, about 125 cm / hr, about 130 cm / hr, about 135 cm / hr, about 140 cm / hr, about 145 cm / hr, about 150 cm / hr, about 155 cm / hr, about 160 cm / hr, about 165 cm / hr, about 170 cm / hr, about 175 cm / hr, about 180 cm / hr, about 185 cm / hr, about 190 cm / hr, about 195 cm / hr, or about 200 cm / hr. Depending on the resin, the flow rate can be up to 600 cm / hr in some embodiments.

[0095] In some embodiments, the flow rate of one or more elution steps of the method is from about 10 cm / hr to about 200 cm / hr, e.g., about 10 cm / hr, about 15 cm / hr, about 20 cm / hr, about 25 cm / hr, about 30 cm / hr, about 35 cm / hr, about 40 cm / hr, about 45 cm / hr, about 50 cm / hr, about 55 cm / hr, about 60 cm / hr, about 65 cm / hr, about 70 cm / hr, about 75 cm / hr, about 80 cm / hr, about 85 cm / hr, about 90 cm / hr, about 95 cm / hr, The flow rate can be about 100 cm / hr, about 105 cm / hr, about 110 cm / hr, about 115 cm / hr, about 120 cm / hr, about 125 cm / hr, about 130 cm / hr, about 135 cm / hr, about 140 cm / hr, about 145 cm / hr, about 150 cm / hr, about 155 cm / hr, about 160 cm / hr, about 165 cm / hr, about 170 cm / hr, about 175 cm / hr, about 180 cm / hr, about 185 cm / hr, about 190 cm / hr, about 195 cm / hr, or about 200 cm / hr. Depending on the resin, the flow rate can be up to 600 cm / hr in some embodiments.

[0096] In some embodiments, the one or more buffers further comprise one or more non-ionic detergents. In some embodiments, the non-ionic detergent is selected from the group consisting of Triton X-100, Tween 80, and Tween 20. In some embodiments, the non-ionic detergent is Triton X-100. In some embodiments, the non-ionic detergent is Tween 80. In some embodiments, the non-ionic detergent is Tween 20.

[0097] In some embodiments, the one or more buffers further comprise one or more additional substances selected from the group consisting of non-reducing sugars, sugar alcohols, and polyols. In some embodiments, the one or more buffers further comprise one or more non-reducing sugars. In some embodiments, non-reducing sugars include, but are not limited to, sucrose, trehalose, mannitol, sorbitol, galactitol, and / or xylitol. In some embodiments, the one or more buffers further comprise one or more sugar alcohols. In some embodiments, the one or more buffers further comprise one or more polyols. In some embodiments, the sugar alcohols or polyols include, but are not limited to, mannitol, xylitol, erythritol, threitol, sorbitol, and / or glycerol. In some embodiments, the buffer further comprises ethylene glycol, propylene glycol, glycerol, 1,2,3-propanetriol), meso-erythritol, and / or erythritol (meso-1,2,3,4-butanetriol).

[0098] In some embodiments, the buffer may include one or more monovalent cations. In some embodiments, the one or more monovalent cations are Na + , K. + , Li + , Cs + , and NH4 + For example, the monovalent cation may be selected from the group consisting of Na +In other embodiments, the buffer comprises one or more monovalent, divalent, and / or trivalent anions. In some embodiments, the one or more monovalent, divalent, and / or trivalent anions are Cl. - , acetic acid - , SO4 2- , Br - , citric acid 3- , PO4 3- , and BO3 3- In some embodiments, the buffer comprises one or more additional substances selected from the group consisting of non-reducing sugars and sugar alcohols. In some embodiments, the one or more buffers further comprise one or more monovalent cations. In some embodiments, the one or more monovalent cations are selected from the group consisting of Na + , K. + , Li + , and Cs + In some embodiments, the monovalent cation is Na+. In some embodiments, the one or more buffers further comprise one or more monovalent, divalent, and / or trivalent anions. In some embodiments, the one or more monovalent, divalent, and / or trivalent anions are selected from the group consisting of Cl - , acetic acid - , SO4 2- , Br - , and citric acid 3- is selected from the group consisting of:

[0099] The pH of any buffer can be adjusted (increased) by adding amino acids, Tris, NaOH, ethanolamine, etc.

[0100] In some embodiments, the anion exchange buffer and chelator combination comprises citrate, malate (malic acid), or tartrate (tartaric acid).

[0101] b. Cation exchange chromatography purification In one embodiment of this method, mature VWF (matVWF) is separated from VWF-PP using cation exchange (CEX) chromatography. In some cases, residual host cell-derived impurities such as CHO host cell proteins, process-related impurities such as recombinant furin and low molecular weight virus inactivation reagents, media compounds such as soy peptone, and other product-related impurities are removed from the mature VWF.

[0102] In another aspect of the method, mature VWF is separated from VWF-PP, such as residual or free VWF-PP, using cation exchange chromatography. For separation, the starting composition, loading solution, or loading composition can contain a low pH and at least one chelating agent. In some embodiments, the starting composition, loading solution, or loading composition contains pro-rVWF, mat-rVWF / rVWF-PP complex, mat-rVWF, and / or rVWF propeptide (rVWF-PP). In some embodiments, the cation exchanger is operated in binding mode, and mature VWF and VWF-PP are separated using a gradient elution buffer containing at least one chelating agent. In other embodiments, the gradient elution buffer has a neutral to high pH, ​​such as a pH in the range of pH 6.0 to pH 9.0. In another embodiment, the gradient elution buffer contains one or more chelating agents and has a pH of 7.0 or higher, e.g., pH 7.0 to pH 9.0. For example, the gradient elution buffer may contain EDTA and have a pH of 8.5.

[0103] In some embodiments, the present invention provides a method for obtaining a composition comprising highly purified mature recombinant rVWF depleted of propeptide (highly purified mat-rVWF), the method comprising the following steps: (a) loading a solution containing pro-rVWF, mat-rVWF / rVWF-PP complex, mat-rVWF, and / or rVWF propeptide (rVWF-PP) onto a cation exchange column to bind the pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF to the cation exchange column; (b) washing the cation exchange column of (a) containing the bound pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF with one or more wash buffers; and (c) treating the column of (b) containing the bound pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF with furin. (d) eluting the bound pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF from the column of (c) with an elution buffer, wherein the elution buffer induces dissociation of the rVWF-PP from mat-rVWF non-covalently associated with the rVWF-PP, and the dissociation is induced by (i) adding at least one chelating agent to the elution buffer or (ii) increasing the pH of the elution buffer to at least pH 7; and (e) separating and recovering the mat-rVWF from the rVWF-PP to obtain a highly purified mat-rVWF composition, wherein the highly purified mat-rVWF composition comprises at least 95% mature rVWF and less than 5% rVWF-PP.

[0104] In some embodiments, a) and b) are performed simultaneously in a single step. In some embodiments, the solution in a) comprises flow-through from an immunoaffinity purification method. In some embodiments, the solution in a) comprises flow-through from a monoclonal antibody column, wherein the monoclonal antibody is a FVIII monoclonal antibody. In some embodiments, the solution in a) is selected from the group consisting of cell culture medium, an antibody column flow-through solution, and a buffer solution.

[0105] The cation exchanger can be operated in binding mode to separate mature VWF and VWF-PP. Cation exchange chromatography can be performed as will be appreciated by those skilled in the art. In some embodiments, the cation exchanger may be POROS® S (Applied Biosystems), Convective Interaction Media (CIM®; BIA Separation), Toyopearl Gigacap S (Tosoh Bioscience, Montgomeryville, PA), Toyopearl Gigacap CM (Tosoh), Toyopearl SP (Tosoha), Toyopearl CM (Tosoh), MacroPrep S (Bio-rad, Hercules, CA), UNOsphere S (Bio-rad, Hercules, CA), Macroprep CM (Bio-rad, Hercules, CA), Fractogel EMD SO (Merck), Fractogel EMD COO (Merck), Fractogel EMD SE Hicap (Merck), Cellfin Sulfate (JNC), CM and SP Trisacryl (Pall), CM and S HyperD (Pall), S and CM Sepharose CL (GE Examples of suitable cation exchangers include, but are not limited to, Sepharose FF (GE Healthcare), S and CM (GE Healthcare), S and CM CAPTO™ (GE Healthcare), MonoS (GE Healthcare), Source S (GE Healthcare), Nuvia S (Merck), or Serfin Phosphate (JNC). In some embodiments, the cation exchanger is a membrane cation exchanger. In some embodiments, membrane ion exchangers include, but are not limited to, Mustang S (Pall) or Sartobind® S. In some embodiments, the cation exchanger is a UNO_Sphere S column (Bio-Rad) or equivalent.

[0106] In some embodiments of step (b), washing the cation exchange column of a) containing the bound pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF uses washing with one or more wash buffers, where the one or more wash buffers include one, two, three, four, and / or five wash buffers. In some embodiments, the second wash buffer contains components for virus inactivation. In some embodiments, when four or five wash buffers are used, the second wash buffer contains components for virus inactivation. In some embodiments, when four or five wash buffers are used, the second or third wash buffer contains components for a virus inactivation treatment. In some embodiments, the virus inactivation treatment is a solvent and detergent (S / D) treatment. In some embodiments, when five wash buffers are used, the first, second, third, and / or fifth wash buffer has a higher pH than the fourth wash buffer. In some embodiments, when five wash buffers are used, the first, second, third, and fifth wash buffers have a pH of about pH 7 to pH 8, and the fourth wash buffer has a pH of about pH 5 to pH 6. In some embodiments, when five wash buffers are used, the first, second, third, and / or fifth wash buffers have a pH of about pH 7.4 to pH 7.5, and the fourth wash buffer has a pH of about pH 5.5. In some embodiments, the virus inactivation step is performed in a buffer having a higher pH than the fourth wash buffer. In some embodiments, when four wash buffers are used, the virus inactivation step is performed after the first wash buffer. In some embodiments, when four wash buffers are used, the first, second, and fourth wash buffers have a higher pH than the third wash buffer. In some embodiments, the virus inactivation step is performed in a buffer having a higher pH than the third wash buffer. In some embodiments, the virus inactivation step is performed in a buffer having the same pH as the first, second, and / or fourth wash buffers. In some embodiments, when four wash buffers are used, the first, second, and fourth wash buffers have a pH of about pH 7 to about pH 8, and the third wash buffer has a pH of about pH 5 to about pH 6.In some embodiments, when four wash buffers are used, the first, second, and fourth wash buffers have a pH of about pH 7.4 to pH 7.5, and the third wash buffer has a pH of about pH 5.5.

[0107] In some embodiments, the loading concentration of pro-VWF is about 90 IU / ml to about 270 IU / ml resin, e.g., about 90 IU / ml to about 270 IU / ml, about 100 IU / ml to about 270 IU / ml, about 110 IU / ml to about 270 IU / ml, about 120 IU / ml to about 270 IU / ml, about 13 ... ml ~ approx. 270IU / ml, approx. 140IU / ml ~ approx. 270IU / ml, approx. 150IU / ml ~ approx. 270IU / ml, approx. 90IU / ml ~ approx. 250IU / ml, approx. 100IU / ml ml ~ approx. 250IU / ml, approx. 110IU / ml ~ approx. 250IU / ml, approx. 120IU / ml ~ approx. 250IU / ml, approx. 130IU / ml ~ approx. 250IU / ml, approx. 130IU / ml ml ~ approx. 250IU / ml, approx. 140IU / ml ~ approx. 250IU / ml, approx. 150IU / ml ~ approx. 250IU / ml, approx. 90IU / ml ~ approx. 200IU / ml, approx. 100IU / ml ml ~ approx. 200IU / ml, approx. 110IU / ml ~ approx. 200IU / ml, approx. 120IU / ml ~ approx. 200IU / ml, approx. 130IU / ml ~ approx. 200IU / ml, approx. 130IU / ml ml to about 200 IU / ml, about 140 IU / ml to about 200 IU / ml, about 150 IU / ml to about 200 IU / ml, about 90 IU / ml to about 100 IU / ml, about 100 IU / ml to about 150 IU / ml, about 150 IU / ml to about 200 IU / ml, about 200 IU / ml to about 250 IU / ml, or about 250 IU / ml to about 270 IU / ml resin.

[0108] In some embodiments, the cation exchange method comprises a buffer system. In some embodiments, the buffer system comprises one or more elution buffers. In some embodiments, the buffer system comprises one or more wash buffers. In some embodiments, the buffer system comprises at least one elution buffer and at least one wash buffer. In some embodiments, the buffer system comprises at least two elution buffers and at least two wash buffers.

[0109] In some embodiments, the first wash buffer comprises at least one chelating agent and optionally has a pH in the range of pH 6.0 to pH 9.0. In some embodiments, the first wash buffer comprises a pH in the range of pH 6.0 to pH 9.0 and optionally comprises at least one chelating agent. In some embodiments, the first wash buffer comprises a pH in the range of pH 6.0 to pH 6.9. In some embodiments, the second wash buffer comprises a pH in the range of pH 7.0 to pH 9.0. In some embodiments, the first wash buffer can comprise at least one chelating agent and has a pH in the range of pH 6.0 to pH 6.9. In some embodiments, the wash elution buffer has a pH less than 7. In one embodiment, the second wash buffer has a pH greater than 7. In some embodiments, when two wash buffers are used, the first wash buffer has a pH less than 7 and the second wash buffer has a pH greater than 7.

[0110] In some embodiments, one or more wash buffers are selected from the group consisting of 120 mM to 200 mM, 130 mM to 200 mM, 140 mM to 200 mM, 150 mM to 200 mM, 120 mM to 190 mM, 130 mM to 190 mM, 140 mM to 190 mM, 150 mM to 190 mM, 120 mM to 180 mM, 130 mM to 180 mM, with NaCl concentrations of 100 mM, 140 mM to 180 mM, 150 mM to 180 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 160 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM, or 200 mM.

[0111] In some embodiments, an initiating composition, loading solution, or loading composition comprising mature VWF and VWF-PP is contacted with a buffer comprising at least one chelating agent, optionally having a pH in the range of pH 6.0 to pH 9.0. In some embodiments, an initiating composition, loading solution, or loading composition is contacted with a buffer comprising at least one chelating agent, optionally having a pH in the range of pH 6.0 to pH 9.0. In some embodiments, an initiating composition, loading solution, or loading composition is contacted with a buffer comprising at least one chelating agent, optionally having a pH in the range of pH 6.0 to pH 9.0. In some embodiments, the buffer is a wash buffer. In some embodiments, the buffer is an elution buffer. In some embodiments, the buffer is a wash buffer having a pH of 6.0 to 6.9. In some embodiments, the buffer is an elution buffer having a pH of 7.0 to 9.0. In some embodiments, an initiating composition, loading solution, or loading composition comprising mature VWF and VWF-PP is contacted first with a wash buffer having a pH of 6.0 to 6.9 and second with at least one elution buffer having a pH of 7.0 to 9.0.

[0112] In some embodiments, mature VWF is eluted in the anion exchange chromatography step using a single elution buffer. In some embodiments, mature VWF is eluted in the anion exchange chromatography step using a gradient elution method comprising multiple elution buffers. For example, elution can be performed using two elution buffers, such as a first elution buffer and a second elution buffer. In some embodiments, the first elution buffer comprises at least one chelating agent and optionally has a pH in the range of pH 6.0 to pH 9.0. In some embodiments, the first elution buffer has a pH in the range of pH 6.0 to pH 9.0 and optionally comprises at least one chelating agent. In some embodiments, the first elution buffer has a pH in the range of pH 6.0 to pH 6.9. In some embodiments, the second elution buffer has a pH in the range of pH 7.0 to pH 9.0. In some embodiments, the first elution buffer can comprise at least one chelating agent and has a pH in the range of pH 6.0 to pH 6.9. In some embodiments, the first elution buffer has a pH less than 7. In one embodiment, the second elution buffer has a pH greater than 7. In some embodiments, when two elution buffers are used, the first elution buffer has a pH less than 7 and the second elution buffer has a pH greater than 7.

[0113] In some embodiments, the pH of the wash buffer for the cation exchange chromatography step is between pH 6.0 and pH 9.0, e.g., pH 6.0 and pH 9.0, pH 6.3 and pH 9.0, pH 6.5 and pH 9.0, pH 7.0 and pH 9.0, pH 7.5 and pH 9.0, pH 7.7.0 and pH 9.0, pH 8.0 and pH 9.0, pH 6.0 and pH 8.5, pH 6.5 and pH 8.5, pH 7.0 and pH 8.5, pH 7.5 and pH 8.5, pH 6.0 and pH 8.0, pH 6.5 and pH 8.0 , pH 7.0 to pH 8.0, pH 7.5 to pH 8.0, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, pH 9.0. In some embodiments, this includes when two elution buffers are present, e.g., first and second elution buffers.

[0114] In some embodiments, the pH of the elution buffer for the cation exchange chromatography step is between pH 6.0 and pH 9.0, e.g., pH 6.0 and pH 9.0, pH 6.3 and pH 9.0, pH 6.5 and pH 9.0, pH 7.0 and pH 9.0, pH 7.5 and pH 9.0, pH 7.7.0 and pH 9.0, pH 8.0 and pH 9.0, pH 6.0 and pH 8.5, pH 6.5 and pH 8.5, pH 7.0 and pH 8.5, pH 7.5 and pH 8.5, pH 6.0 and pH 8.0, pH 6.5 and pH 8.0 , pH 7.0 to pH 8.0, pH 7.5 to pH 8.0, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, pH 9.0. In some embodiments, this includes when two elution buffers are present, e.g., first and second elution buffers.

[0115] In some embodiments, the pH of the elution buffer is increased relative to the starting solution of step a), increased relative to the first elution buffer if two elution buffers are used, and / or increased relative to the wash buffer if a wash buffer is used. In some embodiments, when a wash buffer and an elution buffer are used, the wash buffer has a pH less than 7 and the elution buffer has a pH greater than 7. In some embodiments, when two elution buffers are used, one elution buffer has a pH less than 7 and the other elution buffer has a pH greater than 7. In some embodiments, when a wash buffer and two elution buffers are used, the wash buffer has a pH less than 7 and both elution buffers have a pH greater than 7. In some embodiments, when a wash buffer and two elution buffers are used, the wash buffer and the first elution buffer have a pH less than 7 and the second elution buffer have a pH greater than 7. In some embodiments, when two wash buffers and two elution buffers are used, the wash buffer and the first elution buffer have a pH less than 7 and the second elution buffer have a pH greater than 7. In some embodiments, when two wash buffers and two elution buffers are used, both wash buffers have a pH less than 7 and both elution buffers have a pH greater than 7. In some embodiments, when two wash buffers and two elution buffers are used, the first wash buffer has a pH below 7, and the second wash buffer and both elution buffers have a pH above 7.

[0116] In some embodiments, as described in step (a) of the method, the pH of one or more wash buffers and / or elution buffers is increased to at least 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0 compared to the loading solution containing pro-rVWF, mat-rVWF / rVWF-PP complex, mat-rVWF, and / or rVWF propeptide (rVWF-PP). In some embodiments, to induce dissociation of the mat-rVWF / rVWF-PP complex into mat-rVWF and rVWF-PP in the solution of step (a) of the method, the pH of the buffer is increased to at least 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, where the dissociation occurs by disruption of non-covalently associated mat-rVWF and rVWF-PP. In some embodiments, the pH of the loading solution is increased to at least about 7.2 to about 7.8. In some embodiments, the pH of the loading solution is increased to at least about 7.6. In some embodiments, the pH of the loading solution is increased by the addition of a basic amino acid. In some embodiments, the pH of the loading solution is increased to at least 7. In some embodiments, the pH of one or more wash buffers is increased to at least about 7.2 to about 7.8. In some embodiments, the pH of one or more wash buffers is increased to at least about 7.6. In some embodiments, the pH of one or more wash buffers is increased by the addition of a basic amino acid. In some embodiments, one or more wash buffers exhibit a pH of at least 7. In some embodiments, the pH of one or more elution buffers is increased to at least about 7.2 to about 7.8. In some embodiments, the pH of one or more elution buffers is increased to at least about 7.6. In some embodiments, the pH of one or more elution buffers is increased by the addition of a basic amino acid. In some embodiments, one or more elution buffers exhibit a pH of at least 7.

[0117] In some embodiments, the pH of the loading solution containing pro-rVWF, mat-rVWF / rVWF-PP complex, mat-rVWF, and / or rVWF propeptide (rVWF-PP) is between pH 6.0 and pH 9.0, e.g., pH 6.0 and pH 9.0, pH 6.3 and pH 9.0, pH 6.5 and pH 9.0, pH 7.0 and pH 9.0, pH 7.5 and pH 9.0, pH 7.7.0 and pH 9.0, pH 8.0 and pH 9.0, pH 6.0 and pH 8.5, pH 6.5 and pH 8.5, pH 7.0 and pH 8.5, pH 7.5 and pH 8.5, pH 6.0 to pH 8.0, pH 6.5 to pH 8.0, pH 7.0 to pH 8.0, pH 7.5 to pH 8.0, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, or pH 9.0.

[0118] In some embodiments, the conductivity of the starting composition, loading solution, or loading composition comprising pro-rVWF, mat-rVWF / rVWF-PP complex, mat-rVWF, and / or rVWF propeptide (rVWF-PP) is between about 5 mS / cm and about 40 mS / cm, e.g., between about 5 mS / cm and about 40 mS / cm, between about 10 mS / cm and about 40 mS / cm, between about 15 mS / cm to about 40 mS / cm, about 20 mS / cm to about 40 mS / cm, about 25 mS / cm to about 40 mS / cm, about 30 mS / cm to about 40 mS / cm, about 10 mS / cm to about 40 mS / cm, about 10 mS / cm to about 30 mS / cm, about 5 mS / cm to about 15 mS / cm, about 15 mS / cm to about 30 mS / cm, or about 20 mS / cm to about 40 mS / cm.

[0119] In some embodiments, a loading solution containing pro-rVWF, mat-rVWF / rVWF-PP complex, mat-rVWF, and / or rVWF propeptide (rVWF-PP) is diluted with a buffer containing sodium citrate, for example, but not limited to, 10 mM to 80 mM sodium citrate, 15 mM to 80 mM sodium citrate, 10 mM to 80 mM sodium citrate, 15 mM to 60 mM sodium citrate, 20 mM to 60 mM sodium citrate, 10 mM sodium citrate, 20 mM sodium citrate, 30 mM sodium citrate, 40 mM sodium citrate, 50 mM sodium citrate, 55 mM sodium citrate, 60 mM sodium citrate, 65 mM sodium citrate, 70 mM sodium citrate, 75 mM sodium citrate, 80 mM sodium citrate, etc.

[0120] In some embodiments, the pH of the wash buffer for the cation exchange chromatography step is between pH 6.0 and pH 9.0, e.g., pH 6.0 and pH 9.0, pH 6.3 and pH 9.0, pH 6.5 and pH 9.0, pH 7.0 and pH 9.0, pH 7.5 and pH 9.0, pH 7.7.0 and pH 9.0, pH 8.0 and pH 9.0, pH 6.0 and pH 8.5, pH 6.5 and pH 8.5, pH 7.0 and pH 8.5, pH 7.5 and pH 8.5, pH 6.0 and pH 8.0, pH 6.5 and pH 8.0, pH 7.0 to pH 8.0, pH 7.5 to pH 8.0, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, or pH 9.0.

[0121] In some embodiments, the pH of the elution buffer for the cation exchange chromatography step is between pH 6.0 and pH 9.0, e.g., pH 6.0 and pH 9.0, pH 6.3 and pH 9.0, pH 6.5 and pH 9.0, pH 7.0 and pH 9.0, pH 7.5 and pH 9.0, pH 7.7.0 and pH 9.0, pH 8.0 and pH 9.0, pH 6.0 and pH 8.5, pH 6.5 and pH 8.5, pH 7.0 and pH 8.5, pH 7.5 and pH 8.5, pH 6.0 and pH 8.0, pH 6.5 and pH 8.0, pH 7.0 to pH 8.0, pH 7.5 to pH 8.0, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, or pH 9.0.

[0122] In some embodiments, the pH of the elution buffer is increased relative to the starting solution of step a), increased relative to the first elution buffer if two elution buffers are used, and / or increased relative to the wash buffer if a wash buffer is used. In some embodiments, when a wash buffer and an elution buffer are used, the wash buffer has a pH less than 7 and the elution buffer has a pH greater than 7. In some embodiments, when two elution buffers are used, one elution buffer has a pH less than 7 and the other elution buffer has a pH greater than 7. In some embodiments, when a wash buffer and two elution buffers are used, the wash buffer has a pH less than 7 and both elution buffers have a pH greater than 7. In some embodiments, when a wash buffer and two elution buffers are used, the wash buffer and the first elution buffer have a pH less than 7 and the second elution buffer have a pH greater than 7. In some embodiments, when two wash buffers and two elution buffers are used, the wash buffer and the first elution buffer have a pH less than 7 and the second elution buffer have a pH greater than 7. In some embodiments, when two wash buffers and two elution buffers are used, both wash buffers have a pH less than 7 and both elution buffers have a pH greater than 7. In some embodiments, when two wash buffers and two elution buffers are used, the first wash buffer has a pH below 7, and the second wash buffer and both elution buffers have a pH above 7.

[0123] In some embodiments, as described in step (a) of the method, the pH of one or more wash buffers and / or elution buffers is increased to at least 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0 compared to the loading solution containing pro-rVWF, mat-rVWF / rVWF-PP complex, mat-rVWF, and / or rVWF propeptide (rVWF-PP). In some embodiments, to induce dissociation of the mat-rVWF / rVWF-PP complex into mat-rVWF and rVWF-PP in the solution of step (a) of the method, the pH of the buffer is increased to at least 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, where the dissociation occurs by disruption of non-covalently associated mat-rVWF and rVWF-PP. In some embodiments, the pH of the loading solution is increased to at least about 7.2 to about 7.8. In some embodiments, the pH of the loading solution is increased to at least about 7.6. In some embodiments, the pH of the loading solution is increased by the addition of a basic amino acid. In some embodiments, the pH of the loading solution is increased to at least 7. In some embodiments, the pH of one or more wash buffers is increased to at least about 7.2 to about 7.8. In some embodiments, the pH of one or more wash buffers is increased to at least about 7.6. In some embodiments, the pH of one or more wash buffers is increased by the addition of a basic amino acid. In some embodiments, one or more wash buffers exhibit a pH of at least 7. In some embodiments, the pH of one or more elution buffers is increased to at least about 7.2 to about 7.8. In some embodiments, the pH of one or more elution buffers is increased to at least about 7.6. In some embodiments, the pH of one or more elution buffers is increased by the addition of a basic amino acid. In some embodiments, one or more elution buffers exhibit a pH of at least 7.

[0124] In some embodiments, one or more buffers (including wash buffers and / or elution buffers) comprise one or more chelating agents. In some embodiments, the elution buffer comprises at least one chelating agent. The chelating agent can be a divalent cation chelating agent. In some embodiments, the at least one chelating agent is a divalent cation chelating agent. In some embodiments, the divalent cation chelating agent is selected from the group consisting of EDTA, EGTA, CDTA, and citrate. In some embodiments, the divalent cation chelating agent is selected from the group consisting of NTA, DTPA, EDDS, EDTA, EGTA, CDTA, and citrate. In some embodiments, the divalent cation chelating agent is selected from the group consisting of citrate, EDTA, DTPA, NTA, and EDDS. In some embodiments, the chelating agent is NTA. In some embodiments, the chelating agent is DTPA. In some embodiments, the chelating agent is EDDS. In some embodiments, the chelating agent is EDTA. In some embodiments, the chelating agent is EGTA. In some embodiments, the chelating agent is CDTA. In some embodiments, the chelating agent is citrate. In some embodiments, the one or more wash buffers in b) comprise said one or more chelating agents and exhibit a pH of at least 7.

[0125] Any of the buffers (buffer systems) described herein can be selected from the group consisting of glycine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TrisHCl (tris(hydroxymethyl)-aminomethane), histidine, imidazole, acetate citrate, citrate, acetate, MES, phosphate, TrisHCl, Bis-Tris, histidine, imidazole, arginineHCl, lysineHCl, and 2-(N-morpholino)ethanesulfonic acid, either as a single buffer or as a combination of two or more buffers. In some embodiments, one or more buffers are selected from the group consisting of glycine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TrisHCl (tris(hydroxymethyl)-aminomethane), histidine, imidazole, acetate citrate, MES, and 2-(N-morpholino)ethanesulfonic acid. In some embodiments, the buffer comprises citrate, acetate, MES, HEPES, phosphate, Tris-Cl, and / or Bis-Tris. In some embodiments, the buffer comprises glycine. In some embodiments, the buffer comprises HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). In some embodiments, the buffer comprises Tris-HCl (tris(hydroxymethyl)-aminomethane). In some embodiments, the buffer comprises histidine. In some embodiments, the buffer comprises imidazole. In some embodiments, the buffer comprises acetate-citrate. In some embodiments, the buffer comprises citrate. In some embodiments, the buffer comprises acetate. In some embodiments, the buffer comprises MES. In some embodiments, the buffer comprises HEPES. In some embodiments, the buffer comprises phosphate. In some embodiments, the buffer comprises Tris-HCl. In some embodiments, the buffer comprises Bis-Tris. In some embodiments, the buffer comprises histidine. In some embodiments, the buffer comprises imidazole. In some embodiments, the buffer comprises arginine HCl. In some embodiments, the buffer comprises lysine HCl.In some embodiments, the buffer comprises 2-(N-morpholino)ethanesulfonic acid, hi some embodiments, the buffer comprises one, two, three, or four of the buffers described herein.

[0126] In some embodiments, one or more buffers (including wash and / or elution buffers) comprise sodium citrate in a range including, but not limited to, 10 mM to 80 mM sodium citrate, 15 mM to 80 mM sodium citrate, 10 mM to 80 mM sodium citrate, 15 mM to 60 mM sodium citrate, 20 mM to 60 mM sodium citrate, 10 mM sodium citrate, 20 mM sodium citrate, 30 mM sodium citrate, 40 mM sodium citrate, 50 mM sodium citrate, 55 mM sodium citrate, 60 mM sodium citrate, 65 mM sodium citrate, 70 mM sodium citrate, 75 mM sodium citrate, 80 mM sodium citrate, etc.

[0127] In some embodiments, the first elution buffer further comprises sodium citrate in a range including, but not limited to, 10 mM to 60 mM sodium citrate, 15 mM to 60 mM sodium citrate, 10 mM to 50 mM sodium citrate, 15 mM to 50 mM sodium citrate, 20 mM to 60 mM sodium citrate, 10 mM sodium citrate, 20 mM sodium citrate, 30 mM sodium citrate, 40 mM sodium citrate, 50 mM sodium citrate, 60 mM sodium citrate, etc.

[0128] In some embodiments, the second elution buffer further comprises sodium citrate, for example, but not limited to, 10 mM to 60 mM sodium citrate, 15 mM to 60 mM sodium citrate, 10 mM to 50 mM sodium citrate, 15 mM to 50 mM sodium citrate, 20 mM to 60 mM sodium citrate, 10 mM sodium citrate, 20 mM sodium citrate, 30 mM sodium citrate, 40 mM sodium citrate, 50 mM sodium citrate, 60 mM sodium citrate, etc.

[0129] In some embodiments, one or more buffers (including wash buffers and / or elution buffers) of the cation exchange chromatography step contain EDTA, so long as the desired rVWF species remains bound to the cation exchange resin. In some embodiments, one or more buffers (including wash buffers and / or elution buffers) of the cation exchange chromatography step contain EDTA, so long as the desired rVWF species remains bound to the cation exchange resin. In some embodiments, one or more buffers (including wash buffers and / or elution buffers) of the cation exchange chromatography step contain EDTA, so long as the desired rVWF species remains bound to the cation exchange resin. EDTA, for example, about 0.5mM to about 20mM, about 1mM to about 20mM, about 1.5mM to about 20mM, about 2mM to about 20mM, about 3m M ~ about 20mM, about 5mM - about 20mM, about 0.5mM - about 15mM, about 1mM - about 10mM, about 1mM - about 5mM, about 5mM, about 0. 5mmm, about 1mM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11m M, including about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM, etc. In some embodiments, a buffer containing EDTA is used as part of a stepwise cation exchange elution. In some embodiments, a buffer containing EDTA is used as part of a concentration-gradient cation exchange elution. In some embodiments, when EDTA is used as part of a buffer used in a stepwise cation exchange elution, the counterion is Na+. In some embodiments, when EDTA is used as part of a buffer used in a concentration-gradient cation exchange elution, the counterion is Na+.

[0130] In some embodiments, citrate can be found in the eluate after removal of the rVWF propeptide using a cation exchange method. In some embodiments, citrate can be found in the eluate after removal of the rVWF propeptide using a stepwise cation exchange elution method. In some embodiments, citrate can be found in the eluate after removal of the rVWF propeptide using a gradient cation exchange elution method. In some embodiments, the counterion in the cation exchange is Na + is.

[0131] In some embodiments, the conductivity of the buffer (including wash buffer and / or elution buffer) is in the range of 5 mS / cm to 40 mS / cm, e.g., 5 mS / cm to 40 mS / cm, 10 mS / cm to 40 mS / cm, 15 mS / cm to 40 mS / cm, 20 mS / cm to 40 mS / cm, 5 mS / cm to 15 mS / cm, 10 mS / cm to 25 mS / cm, 15 mS / cm to 30 mS / cm, 20 mS / cm to 30 mS / cm, or 30 mS / cm to 40 mS / cm.

[0132] In some embodiments, the conductivity of at least one wash buffer is from about 5 mS / cm to about 40 mS / cm, e.g., from about 5 mS / cm to about 40 mS / cm, from about 10 mS / cm to about 40 mS / cm, from about 15 mS / cm to about 40 mS / cm, from about 20 mS / cm to about 40 mS / cm, from about 25 mS / cm to about 40 mS / cm, from about 30 mS / cm to about 40 mS / cm, from about 10 mS / cm to about 40 mS / cm, from about 10 mS / cm to about 30 mS / cm, from about 5 mS / cm to about 13 mS / cm, from about 5 mS / cm to about 15 mS / cm, from about 15 mS / cm to about 30 mS / cm, from about 18 mS / cm to about 40 mS / cm, or from about 20 mS / cm to about 40 mS / cm. In other embodiments, the conductivity of the two or more wash buffer solutions is from about 5 mS / cm to about 40 mS / cm, e.g., from about 5 mS / cm to about 40 mS / cm, from about 10 mS / cm to about 40 mS / cm, from about 15 mS / cm to about 40 mS / cm, from about 20 mS / cm to about 40 mS / cm, from about 25 mS / cm to about 40 mS / cm, from about 30 mS / cm to about 40 mS / cm, from about 10 mS / cm to about 40 mS / cm, from about 10 mS / cm to about 30 mS / cm, from about 5 mS / cm to about 13 mS / cm, from about 5 mS / cm to about 15 mS / cm, from about 15 mS / cm to about 30 mS / cm, from about 18 mS / cm to about 40 mS / cm, or from about 20 mS / cm to about 40 mS / cm.

[0133] In some embodiments, the conductivity of at least one elution buffer is from about 5 mS / cm to about 40 mS / cm, e.g., from about 5 mS / cm to about 40 mS / cm, from about 10 mS / cm to about 40 mS / cm, from about 15 mS / cm to about 40 mS / cm, from about 20 mS / cm to about 40 mS / cm, from about 25 mS / cm to about 40 mS / cm, from about 30 mS / cm to about 40 mS / cm, from about 10 mS / cm to about 40 mS / cm, from about 10 mS / cm to about 30 mS / cm, from about 5 mS / cm to about 13 mS / cm, from about 5 mS / cm to about 15 mS / cm, from about 15 mS / cm to about 30 mS / cm, from about 18 mS / cm to about 40 mS / cm, or from about 20 mS / cm to about 40 mS / cm. In other embodiments, the conductivity of the two or more wash buffer solutions is from about 5 mS / cm to about 40 mS / cm, e.g., from about 5 mS / cm to about 40 mS / cm, from about 10 mS / cm to about 40 mS / cm, from about 15 mS / cm to about 40 mS / cm, from about 20 mS / cm to about 40 mS / cm, from about 25 mS / cm to about 40 mS / cm, from about 30 mS / cm to about 40 mS / cm, from about 10 mS / cm to about 40 mS / cm, from about 10 mS / cm to about 30 mS / cm, from about 5 mS / cm to about 13 mS / cm, from about 5 mS / cm to about 15 mS / cm, from about 15 mS / cm to about 30 mS / cm, from about 18 mS / cm to about 40 mS / cm, or from about 20 mS / cm to about 40 mS / cm.

[0134] In some embodiments, the pH of the wash buffer is between pH 6.0 and pH 9.0, e.g., pH 6.0 and pH 9.0, pH 6.3 and pH 9.0, pH 6.5 and pH 9.0, pH 7.0 and pH 9.0, pH 7.5 and pH 9.0, pH 7.7.0 and pH 9.0, pH 8.0 and pH 9.0, pH 6.0 and pH 8.5, pH 6.5 and pH 8.5, pH 7.0 and pH 8.5, pH 7.5 and pH 8.5, pH 6.0 and pH 8.0, pH 6.5 and pH 8.0, pH 7.0 and pH 8.0, pH7.5~pH8.0, pH6.0, pH6.1, pH6.2, pH6.3, pH6.4, pH6.5, pH6.6, pH6.7, pH6.8, pH6.9, pH7.0, pH7.1, pH7.2, pH7.3, pH7.4, pH7.5, pH7.6, pH7.7, pH7.8, pH7.9, pH8.0, pH8.1, pH8.2, pH8.3, pH8.4, pH8.5, pH8.6, pH8.7, pH8.8, pH8.9, pH9.0.

[0135] In one embodiment, the methods described herein include a gradient elution step, which can remove product impurities and process-related impurities to optimize the yield of mature VWF. In some cases, the gradient elution step separates a higher proportion of VWF propeptides from mature VWF than conventional methods.

[0136] In some embodiments, the conductivity of the one or more elution buffers is from about 5 mS / cm to about 40 mS / cm, e.g., from about 5 mS / cm to about 40 mS / cm, from about 10 mS / cm to about 40 mS / cm, from about 15 mS / cm to about 40 mS / cm, from about 20 mS / cm to about 40 mS / cm, from about 25 mS / cm to about 40 mS / cm, from about 30 mS / cm to about 40 mS / cm, from about 10 mS / cm to about 40 mS / cm, from about 10 mS / cm to about 30 mS / cm, from about 5 mS / cm to about 13 mS / cm, from about 5 mS / cm to about 15 mS / cm, from about 15 mS / cm to about 30 mS / cm, from about 18 mS / cm to about 40 mS / cm, or from about 20 mS / cm to about 40 mS / cm. In other embodiments, the conductivity of the two or more wash buffer solutions is from about 5 mS / cm to about 40 mS / cm, e.g., from about 5 mS / cm to about 40 mS / cm, from about 10 mS / cm to about 40 mS / cm, from about 15 mS / cm to about 40 mS / cm, from about 20 mS / cm to about 40 mS / cm, from about 25 mS / cm to about 40 mS / cm, from about 30 mS / cm to about 40 mS / cm, from about 10 mS / cm to about 40 mS / cm, from about 10 mS / cm to about 30 mS / cm, from about 5 mS / cm to about 13 mS / cm, from about 5 mS / cm to about 15 mS / cm, from about 15 mS / cm to about 30 mS / cm, from about 18 mS / cm to about 40 mS / cm, or from about 20 mS / cm to about 40 mS / cm.

[0137] In some embodiments, the flow rate of one or more wash steps of the method is from about 10 cm / hr to about 200 cm / hr, e.g., about 10 cm / hr, about 15 cm / hr, about 20 cm / hr, about 25 cm / hr, about 30 cm / hr, about 35 cm / hr, about 40 cm / hr, about 45 cm / hr, about 50 cm / hr, about 55 cm / hr, about 60 cm / hr, about 65 cm / hr, about 70 cm / hr, about 75 cm / hr, about 80 cm / hr, about 85 cm / hr, about 90 cm / hr, about 95 cm / hr, The flow rate can be about 100 cm / hr, about 105 cm / hr, about 110 cm / hr, about 115 cm / hr, about 120 cm / hr, about 125 cm / hr, about 130 cm / hr, about 135 cm / hr, about 140 cm / hr, about 145 cm / hr, about 150 cm / hr, about 155 cm / hr, about 160 cm / hr, about 165 cm / hr, about 170 cm / hr, about 175 cm / hr, about 180 cm / hr, about 185 cm / hr, about 190 cm / hr, about 195 cm / hr, or about 200 cm / hr. Depending on the resin, the flow rate can be up to 600 cm / hr in some embodiments.

[0138] In some embodiments, the flow rate of one or more elution steps of the method is from about 10 cm / hr to about 200 cm / hr, e.g., about 10 cm / hr, about 15 cm / hr, about 20 cm / hr, about 25 cm / hr, about 30 cm / hr, about 35 cm / hr, about 40 cm / hr, about 45 cm / hr, about 50 cm / hr, about 55 cm / hr, about 60 cm / hr, about 65 cm / hr, about 70 cm / hr, about 75 cm / hr, about 80 cm / hr, about 85 cm / hr, about 90 cm / hr, about 95 cm / hr, The flow rate can be about 100 cm / hr, about 105 cm / hr, about 110 cm / hr, about 115 cm / hr, about 120 cm / hr, about 125 cm / hr, about 130 cm / hr, about 135 cm / hr, about 140 cm / hr, about 145 cm / hr, about 150 cm / hr, about 155 cm / hr, about 160 cm / hr, about 165 cm / hr, about 170 cm / hr, about 175 cm / hr, about 180 cm / hr, about 185 cm / hr, about 190 cm / hr, about 195 cm / hr, or about 200 cm / hr. Depending on the resin, the flow rate can be up to 600 cm / hr in some embodiments.

[0139] In some embodiments, the one or more buffers further comprise one or more non-ionic detergents. In some embodiments, the non-ionic detergent is selected from the group consisting of Triton X-100, Tween 80, and Tween 20. In some embodiments, the non-ionic detergent is Triton X-100. In some embodiments, the non-ionic detergent is Tween 80. In some embodiments, the non-ionic detergent is Tween 20.

[0140] In some embodiments, the one or more buffers further comprise one or more additional substances selected from the group consisting of non-reducing sugars, sugar alcohols, and polyols. In some embodiments, the one or more buffers further comprise one or more non-reducing sugars. In some embodiments, non-reducing sugars include, but are not limited to, sucrose, trehalose, mannitol, sorbitol, galactitol, and / or xylitol. In some embodiments, the one or more buffers further comprise one or more sugar alcohols. In some embodiments, the one or more buffers further comprise one or more polyols. In some embodiments, the sugar alcohols or polyols include, but are not limited to, mannitol, xylitol, erythritol, threitol, sorbitol, and / or glycerol. In some embodiments, the buffer further comprises sorbitol, mannitol, xylitol, sucrose, trehalose, ethylene glycol, propylene glycol, glycerol, 1,2,3-propanetriol, meso-erythritol, and / or erythritol (meso-1,2,3,4-butanetriol).

[0141] The pH of any buffer can be adjusted (increased) by adding amino acids, Tris, NaOH, ethanolamine, etc.

[0142] Any of the buffers (buffer systems) described herein, either as a single buffer or as a combination of two or more buffers, can be selected from the group consisting of citrate, acetate, MES, HEPES, phosphate, Tris-Cl, and Bis-Tris. In some embodiments, the buffer comprises glycine. In some embodiments, the buffer comprises citrate. In some embodiments, the buffer comprises acetate. In some embodiments, the buffer comprises MES. In some embodiments, the buffer comprises HEPES. In some embodiments, the buffer comprises phosphate. In some embodiments, the buffer comprises Tris-HCl. In some embodiments, the buffer comprises Bis-Tris. In some embodiments, the buffer comprises one, two, three, or four of the buffers described herein.

[0143] In some embodiments, the cation exchange buffer and chelator combination comprises citrate, malate (malic acid), or tartrate (tartaric acid).

[0144] c. Size exclusion chromatography purification In one embodiment of the invention, mature VWF and VWF-PP are separated by size exclusion chromatography (SEC). In some cases, residual host cell-derived impurities such as CHO host cell proteins, process-related impurities such as recombinant furin and low molecular weight virus inactivation reagents, media compounds such as soy peptone, and other product-related impurities are removed from the mature VWF.

[0145] In another aspect of the method, mature VWF is separated from VWF-PP, such as residual or free VWF-PP, using size exclusion chromatography. For separation, the starting or loading composition can contain a low pH and at least one chelating agent. In another embodiment, the gradient elution buffer has a neutral to high pH, ​​such as a pH in the range of pH 6.0 to pH 9.0. In another embodiment, the gradient elution buffer contains one or more chelating agents and has a pH of 7.0 or higher, e.g., pH 7.0 to pH 9.0. For example, the gradient elution buffer can contain EDTA and have a pH of 8.5.

[0146] In some embodiments, the present invention provides a method for obtaining a composition comprising highly purified mature recombinant rVWF depleted of propeptide (highly purified mat-rVWF), the method comprising the following steps: (a) loading a solution containing pro-rVWF, mat-rVWF / rVWF-PP complex, mat-rVWF, and / or rVWF propeptide (rVWF-PP) onto a size-exclusion column and allowing the pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF to bind to the size-exclusion column; (b) washing the size-exclusion column of (a) containing the bound pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF with one or more wash buffers; and (c) treating the column of (b) containing the bound pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF with furin. (d) eluting the bound pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF from the column of (c) with an elution buffer, wherein the elution buffer induces dissociation of the rVWF-PP from mat-rVWF non-covalently associated with the rVWF-PP, the dissociation being induced by (i) adding at least one chelating agent to the elution buffer or (ii) increasing the pH of the elution buffer to at least pH 7; and (e) separating and recovering the mat-rVWF from the rVWF-PP to obtain a highly purified mat-rVWF composition, wherein the highly purified mat-rVWF composition comprises at least 95% mature rVWF and less than 5% rVWF-PP.

[0147] In some embodiments, a) and b) are performed simultaneously in a single step. In some embodiments, the solution in a) comprises flow-through from an immunoaffinity purification method. In some embodiments, the solution in a) comprises flow-through from a monoclonal antibody column, wherein the monoclonal antibody is a FVIII monoclonal antibody. In some embodiments, the solution in a) is selected from the group consisting of cell culture medium, an antibody column flow-through solution, and a buffer solution.

[0148] In some embodiments, the separation buffer has a neutral to high pH. In other embodiments, the buffer contains at least one chelating agent. In some embodiments, the buffer contains at least one chelating agent and has a neutral to high pH. For example, the separation buffer may contain a chelating agent and have a pH of 6.0 or higher, or in some cases, a pH of 7.0 or higher.

[0149] In some embodiments, the loading concentration of pro-VWF is about 90 IU / ml to about 270 IU / ml resin, e.g., about 90 IU / ml to about 270 IU / ml, about 100 IU / ml to about 270 IU / ml, about 110 IU / ml to about 270 IU / ml, about 120 IU / ml to about 270 IU / ml, about 13 ... ml ~ approx. 270IU / ml, approx. 140IU / ml ~ approx. 270IU / ml, approx. 150IU / ml ~ approx. 270IU / ml, approx. 90IU / ml ~ approx. 250IU / ml, approx. 100IU / ml ml ~ approx. 250IU / ml, approx. 110IU / ml ~ approx. 250IU / ml, approx. 120IU / ml ~ approx. 250IU / ml, approx. 130IU / ml ~ approx. 250IU / ml, approx. 130IU / ml ml ~ approx. 250IU / ml, approx. 140IU / ml ~ approx. 250IU / ml, approx. 150IU / ml ~ approx. 250IU / ml, approx. 90IU / ml ~ approx. 200IU / ml, approx. 100IU / ml ml ~ approx. 200IU / ml, approx. 110IU / ml ~ approx. 200IU / ml, approx. 120IU / ml ~ approx. 200IU / ml, approx. 130IU / ml ~ approx. 200IU / ml, approx. 130IU / ml ml to about 200 IU / ml, about 140 IU / ml to about 200 IU / ml, about 150 IU / ml to about 200 IU / ml, about 90 IU / ml to about 100 IU / ml, about 100 IU / ml to about 150 IU / ml, about 150 IU / ml to about 200 IU / ml, about 200 IU / ml to about 250 IU / ml, or about 250 IU / ml to about 270 IU / ml resin.

[0150] In some embodiments, the pH of the starting composition, loading solution, or loading composition comprising pro-rVWF, mat-rVWF / rVWF-PP complex, mat-rVWF, and / or rVWF propeptide (rVWF-PP) is between pH 6.0 and pH 9.0, e.g., pH 6.0 and pH 9.0, pH 6.3 and pH 9.0, pH 6.5 and pH 9.0, pH 7.0 and pH 9.0, pH 7.5 and pH 9.0, pH 7.7 and pH 9.0, pH 8.0 and pH 9.0, pH 6.0 and pH 8.5, pH 6.5 and pH 8.5, pH 7.0 ... .5 to pH8.5, pH6.0 to pH8.0, pH6.5 to pH8.0, pH7.0 to pH8.0, pH7.5 to pH8.0, pH6.0, pH6.1, pH6.2, pH6.3, pH6.4, pH6.5, pH6.6, pH6.7, pH6.8, pH6.9, pH7.0, pH7.1, pH7.2, pH7.3, pH7.4, pH7.5, pH7.6, pH7.7, pH7.8, pH7.9, pH8.0, pH8.1, pH8.2, pH8.3, pH8.4, pH8.5, pH8.6, pH8.7, pH8.8, pH8.9, or pH9.0.

[0151] In some embodiments, the size exclusion method comprises a buffer system. In some embodiments, the buffer system comprises one or more separation buffers. In some embodiments, the buffer system comprises at least one separation buffer. In some embodiments, the buffer system comprises at least two separation buffers. In some embodiments, the buffer system comprises at least a first separation buffer and at least a second separation buffer.

[0152] In some embodiments, the first separation buffer comprises at least one chelating agent and optionally has a pH in the range of pH 6.0 to pH 9.0. In some embodiments, the separation wash buffer has a pH in the range of pH 6.0 to pH 9.0 and optionally comprises at least one chelating agent. In some embodiments, the first separation buffer has a pH in the range of pH 6.0 to pH 6.9. In some embodiments, the second separation buffer has a pH in the range of pH 7.0 to pH 9.0. In some embodiments, the first separation buffer can comprise at least one chelating agent and has a pH in the range of pH 6.0 to pH 6.9. In some embodiments, the first separation buffer has a pH less than 7. In some embodiments, the second separation buffer has a pH greater than 7. In some embodiments, when two separation buffers are used, the first separation buffer has a pH less than 7 and the second separation buffer has a pH greater than 7.

[0153] In some embodiments, a starting solution containing mature rVWF and rVWF-PP is contacted with a separation buffer containing at least one chelating agent, and optionally the buffer has a pH in the range of pH 6.0 to pH 9.0. In some embodiments, the starting solution is contacted with a buffer having a pH in the range of pH 6.0 to pH 9.0, and optionally the buffer contains at least one chelating agent. In some embodiments, the buffer has a pH in the range of pH 7.0 to pH 9.0. In some embodiments, the buffer is a first separation buffer having a pH of 6.0 to 6.9. In some embodiments, the buffer is a second separation buffer having a pH of 7.0 to 9.0. In some embodiments, a starting solution containing mature rVWF and rVWF-PP is first contacted with a first buffer having a pH of 6.0 to 6.9 and a second separation buffer having a pH of 7.0 to 9.0.

[0154] In some embodiments, the pH of one or more separation buffers is between pH 6.0 and pH 9.0, e.g., pH 6.0 and pH 9.0, pH 6.3 and pH 9.0, pH 6.5 and pH 9.0, pH 7.0 and pH 9.0, pH 7.5 and pH 9.0, pH 7.7.0 and pH 9.0, pH 8.0 and pH 9.0, pH 6.0 and pH 8.5, pH 6.5 and pH 8.5, pH 7.0 and pH 8.5, pH 7.5 and pH 8.5, pH 6.0 and pH 8.0, pH 6.5 and pH 8.0, pH 7.0 and pH 8.0, pH 7.5~pH 8.0, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6, pH 8.7, pH 8.8, pH 8.9, pH 9.0.

[0155] In some embodiments, the pH of the elution buffer is increased relative to the starting solution of step a), increased relative to the first separation buffer if two separation buffers are used, and / or increased relative to the first separation buffer if a second separation buffer is used. In some embodiments, when a first and a second separation buffer are used, the first separation buffer has a pH below 7 and the second separation buffer has a pH above 7.

[0156] In some embodiments, the pH of one or more separation buffers is increased to at least 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0 relative to the loading solution containing pro-rVWF, mat-rVWF / rVWF-PP complex, mat-rVWF, and / or rVWF propeptide (rVWF-PP) as described in step (a) of the method. In some embodiments, the pH of the buffer is increased to at least 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0 to induce dissociation of the mat-rVWF / rVWF-PP complex into mat-rVWF and rVWF-PP in the solution of step (a) of the method, wherein the dissociation occurs by disruption of non-covalently associated mat-rVWF and rVWF-PP. In some embodiments, the pH of the loading solution is increased to at least about 7.2 to about 7.8. In some embodiments, the pH of the loading solution is increased to at least about 7.6. In some embodiments, the pH of the loading solution is increased by the addition of a basic amino acid. In some embodiments, the pH of the loading solution is increased to at least 7. In some embodiments, the pH of one or more wash buffers is increased to at least about 7.2 to about 7.8. In some embodiments, the pH of one or more wash buffers is increased to at least about 7.6. In some embodiments, the pH of one or more separation buffers is increased by the addition of a basic amino acid. In some embodiments, one or more separation buffers exhibit a pH of at least 7.

[0157] In some embodiments, the one or more separation buffers comprise one or more chelating agents. In some embodiments, the elution buffer comprises at least one chelating agent. The chelating agent can be a divalent cation chelating agent. In some embodiments, the at least one chelating agent is a divalent cation chelating agent. In some embodiments, the divalent cation chelating agent is selected from the group consisting of EDTA, EGTA, CDTA, and citrate. In some embodiments, the divalent cation chelating agent is selected from the group consisting of NTA, DTPA, EDDS, EDTA, EGTA, CDTA, and citrate. In some embodiments, the chelating agent is NTA. In some embodiments, the chelating agent is DTPA. In some embodiments, the chelating agent is EDDS. In some embodiments, the chelating agent is EDTA. In some embodiments, the chelating agent is EGTA. In some embodiments, the chelating agent is CDTA. In some embodiments, the chelating agent is citrate. In some embodiments, the one or more wash buffers in b) comprise the one or more chelating agents and exhibit a pH of at least 7.

[0158] In some embodiments, one or more separation buffers comprise at least one chelating agent. The chelating agent can be a divalent cation chelating agent. In some embodiments, the divalent cation chelating agent is selected from the group consisting of nitrilo-2,2',2"-triacetic acid (NTA), diethylenetriaminepentaacetic acid; diethylenetriamine-N,N,N',N',N"pentaacetic acid (DTPA), ethylenediamine-N,N'-disuccinic acid (EDDS), ethylenediaminetetraacetic acid (EDTA), EGTA, CDTA, and citrate. In some embodiments, the divalent cation chelating agent is selected from the group consisting of NTA, DTPA, EDDS, EDTA, and citrate. In some embodiments, the chelating agent is NTA. In some embodiments, the chelating agent is DTPA. In some embodiments, the chelating agent is EDDS. In some embodiments, the chelating agent is EDTA. In some embodiments, the chelating agent is EGTA. In some embodiments, the chelating agent is CDTA. In some embodiments, the chelating agent is citrate.

[0159] In some embodiments, elution buffer A and / or elution buffer B in the anion exchange chromatography step may be from about 0.5 mM to about 20 mM EDTA, for example, about 0.5mM to about 20mM, about 1mM to about 20mM, about 1.5mM to about 20mM, about 2mM to about 20mM, about 3m M ~ about 20mM, about 5mM - about 20mM, about 0.5mM - about 15mM, about 1mM - about 10mM, about 1mM - about 5mM, about 5mM, about 0. 5mmm, about 1mM, about 2mM, about 3mM, about 4mM, about 5mM, about 6mM, about 7mM, about 8mM, about 9mM, about 10mM, about 11m M, including about 12mM, about 13mM, about 14mM, about 15mM, about 16mM, about 17mM, about 18mM, about 19mM, about 20mM, etc.

[0160] In some embodiments, the one or more separation buffers are selected from the group consisting of 10 mM to 500 mM sodium citrate, 15 mM to 400 mM sodium citrate, 10 mM to 400 mM sodium citrate, 15 mM to 350 mM sodium citrate, 20 mM to 350 mM sodium citrate, 10 mM sodium citrate, 20 mM sodium citrate, 30 mM sodium citrate, 40 mM sodium citrate, 50 mM sodium citrate, 55 mM sodium citrate, 60 mM sodium citrate, 65 mM sodium citrate, 70 mM sodium citrate , 75mM sodium citrate, 80mM sodium citrate, 90mM sodium citrate, 100mM sodium citrate, 110mM sodium citrate, 120mM sodium citrate, 130mM sodium citrate, 140mM sodium citrate, 150mM sodium citrate, 160mM sodium citrate, 170mM sodium citrate, 180mM sodium citrate, 190mM sodium citrate, 200mM sodium citrate, 210mM ​​sodium citrate, 220mM sodium citrate, 230mM sodium citrate Sodium, 240mM sodium citrate, 250mM sodium citrate, 260mM sodium citrate, 270mM sodium citrate, 280mM sodium citrate, 290mM sodium citrate, 300mM sodium citrate, 310mM sodium citrate, 320mM sodium citrate, 330mM sodium citrate, 340mM sodium citrate, 350mM sodium citrate, 360mM sodium citrate, 370mM sodium citrate, 380mM sodium citrate, 390mM sodium citrate, 400mM Sodium citrate, 410mM sodium citrate, 420mM sodium citrate, 430mM sodium citrate, 440mM sodium citrate, 450mM sodium citrate, 460mM sodium citrate, 470mM sodium citrate, 480mM sodium citrate, 490mM sodium citrate, 500mM sodium citrate, 510mM sodium citrate, 520mM sodium citrate, 530mM sodium citrate, 540mM sodium citrate, 550mM sodium citrate, 560mM sodium citrate,The sodium citrate range includes, but is not limited to, 570 mM sodium citrate, 580 mM sodium citrate, 590 mM sodium citrate, or 600 mM sodium citrate.

[0161] In some embodiments, the one or more separation buffers are selected from the group consisting of 10 mM to 500 mM sodium citrate, 15 mM to 400 mM sodium citrate, 10 mM to 400 mM sodium citrate, 15 mM to 350 mM sodium citrate, 20 mM to 350 mM sodium citrate, 10 mM sodium citrate, 20 mM sodium citrate, 30 mM sodium citrate, 40 mM sodium citrate, 50 mM sodium citrate, 55 mM sodium citrate, 60 mM sodium citrate, 65 mM sodium citrate, 70 mM sodium citrate , 75mM sodium citrate, 80mM sodium citrate, 90mM sodium citrate, 100mM sodium citrate, 110mM sodium citrate, 120mM sodium citrate, 130mM sodium citrate, 140mM sodium citrate, 150mM sodium citrate, 160mM sodium citrate, 170mM sodium citrate, 180mM sodium citrate, 190mM sodium citrate, 200mM sodium citrate, 210mM ​​sodium citrate, 220mM sodium citrate, 230mM sodium citrate Sodium, 240mM sodium citrate, 250mM sodium citrate, 260mM sodium citrate, 270mM sodium citrate, 280mM sodium citrate, 290mM sodium citrate, 300mM sodium citrate, 310mM sodium citrate, 320mM sodium citrate, 330mM sodium citrate, 340mM sodium citrate, 350mM sodium citrate, 360mM sodium citrate, 370mM sodium citrate, 380mM sodium citrate, 390mM sodium citrate, 400mM Sodium citrate, 410mM sodium citrate, 420mM sodium citrate, 430mM sodium citrate, 440mM sodium citrate, 450mM sodium citrate, 460mM sodium citrate, 470mM sodium citrate, 480mM sodium citrate, 490mM sodium citrate, 500mM sodium citrate, 510mM sodium citrate, 520mM sodium citrate, 530mM sodium citrate, 540mM sodium citrate, 550mM sodium citrate, 560mM sodium citrate,Further included are sodium citrate ranges including, but not limited to, 570 mM sodium citrate, 580 mM sodium citrate, 590 mM sodium citrate, or 600 mM sodium citrate.

[0162] In some embodiments, one or more separation buffers contain sodium citrate, for example, but not limited to, 10 mM to 500 mM sodium citrate, 15 mM to 400 mM sodium citrate, 10 mM to 400 mM sodium citrate, 15 mM to 350 mM sodium citrate, 20 mM to 350 mM sodium citrate, 10 mM sodium citrate, 20 mM sodium citrate, 30 mM sodium citrate, 40 mM sodium citrate, 50 mM sodium citrate, 55 mM sodium citrate, 60 mM sodium citrate, 65 mM sodium citrate, 70 mM sodium citrate, 75 mM sodium citrate, 80 mM sodium citrate, 85 mM sodium citrate, 90 mM sodium citrate, 95 mM sodium citrate, 10 ... M sodium citrate, 70mM sodium citrate, 75mM sodium citrate, 80mM sodium citrate, 90mM sodium citrate, 100mM sodium citrate, 110mM sodium citrate, 120mM sodium citrate, 130mM sodium citrate, 140mM sodium citrate, 150mM sodium citrate, 160mM sodium citrate, 170mM sodium citrate, 180mM sodium citrate, 190mM sodium citrate, 200mM sodium citrate, 210mM ​​sodium citrate, 220mM sodium citrate, 230mM sodium citrate, 240mM sodium citrate, 250mM sodium citrate, 260mM sodium citrate, 270mM sodium citrate, 280mM sodium citrate, 290mM sodium citrate, 300mM sodium citrate, 310mM sodium citrate, 320mM sodium citrate, 330mM sodium citrate, 340mM sodium citrate, 350mM sodium citrate, 360mM sodium citrate, 370mM sodium citrate, 380mM sodium citrate Sodium citrate, 390mM sodium citrate, 400mM sodium citrate, 410mM sodium citrate, 420mM sodium citrate, 430mM sodium citrate, 440mM sodium citrate, 450mM sodium citrate, 460mM sodium citrate, 470mM sodium citrate, 480mM sodium citrate, 490mM sodium citrate, 500mM sodium citrate, 510mM sodium citrate, 520mM sodium citrate, 530mM sodium citrate, 540mM sodium citrate,Further containing 550 mM sodium citrate, 560 mM sodium citrate, 570 mM sodium citrate, 580 mM sodium citrate, 590 mM sodium citrate, or 600 mM sodium citrate.

[0163] In some embodiments, the conductivity of the separation buffer is about 5 mS / cm to about 40 mS / cm, e.g., about 5 mS / cm to about 40 mS / cm, about 10 mS / cm to about 40 mS / cm, about 15 mS / cm to about 40 mS / cm, about 20 mS / cm to about 40 mS / cm, about 25 mS / cm to about 40 mS / cm, about 30 mS / cm to about 40 mS / cm, about 10 mS / cm to about 40 mS / cm, about 10 mS / cm to about 30 mS / cm, about 5 mS / cm to about 13 mS / cm, about 5 mS / cm to about 15 mS / cm, about 15 mS / cm to about 30 mS / cm, about 18 mS / cm to about 40 mS / cm, or about 20 mS / cm to about 40 mS / cm.

[0164] Any of the buffers (buffer systems) described herein, either as a single buffer or as a combination of two or more buffers, can be selected from the group consisting of citrate, acetate, MES, HEPES, phosphate, Tris-HCl, Bis-Tris, histidine, imidazole, arginine HCl, lysine HCl, glycine, glycylglycine, borate, MOPS, bicine, tricine, TAPS, TAPSO, and PIPES. In some embodiments, the buffer comprises glycine. In some embodiments, the buffer comprises citrate, acetate, MES, HEPES, phosphate, Tris-HCl, Bis-Tris, histidine, imidazole, arginine HCl, lysine HCl, glycine, glycylglycine, borate, MOPS, bicine, tricine, TAPS, TAPSO, and / or PIPES. In some embodiments, the buffer comprises citrate. In some embodiments, the buffer comprises acetate. In some embodiments, the buffer comprises MES. In some embodiments, the buffer comprises HEPES. In some embodiments, the buffer comprises phosphate, in some embodiments, the buffer comprises Tris-HCl, in some embodiments, the buffer comprises Bis-Tris.

[0165] In some embodiments, the buffer comprises histidine. In some embodiments, the buffer comprises imidazole. In some embodiments, the buffer comprises arginine HCl. In some embodiments, the buffer comprises lysine HCl. In some embodiments, the buffer comprises glycine. In some embodiments, the buffer comprises glycylglycine. In some embodiments, the buffer comprises borate. In some embodiments, the buffer comprises MOPS. In some embodiments, the buffer comprises bicine. In some embodiments, the buffer comprises tricine. In some embodiments, the buffer comprises TAPS. In some embodiments, the buffer comprises TAPSO. In some embodiments, the buffer comprises PIPES. In some embodiments, the buffer comprises one, two, three, or four of the buffers described herein.

[0166] In some embodiments, the one or more separation buffers further comprise one or more non-ionic detergents. In some embodiments, the non-ionic detergent is selected from the group consisting of Triton X-100, Tween 80, and Tween 20. In some embodiments, the non-ionic detergent is Triton X-100. In some embodiments, the non-ionic detergent is Tween 80. In some embodiments, the non-ionic detergent is Tween 20.

[0167] In some embodiments, the use of flow rates during one or more separation buffer steps of the method is between about 10 cm / hr and about 200 cm / hr, e.g., about 10 cm / hr, about 15 cm / hr, about 20 cm / hr, about 25 cm / hr, about 30 cm / hr, about 35 cm / hr, about 40 cm / hr, about 45 cm / hr, about 50 cm / hr, about 55 cm / hr, about 60 cm / hr, about 65 cm / hr, about 70 cm / hr, about 75 cm / hr, about 80 cm / hr, about 85 cm / hr, about 90 cm / hr, about 95 cm / hr / hr, about 100 cm / hr, about 105 cm / hr, about 110 cm / hr, about 115 cm / hr, about 120 cm / hr, about 125 cm / hr, about 130 cm / hr, about 135 cm / hr, about 140 cm / hr, about 145 cm / hr, about 150 cm / hr, about 155 cm / hr, about 160 cm / hr, about 165 cm / hr, about 170 cm / hr, about 175 cm / hr, about 180 cm / hr, about 185 cm / hr, about 190 cm / hr, about 195 cm / hr, or about 200 cm / hr. Depending on the resin, the flow rate can be up to 600 cm / hr in some embodiments.

[0168] In some embodiments, the use of flow rates during one or more separation buffer steps of the method is between about 10 cm / hr and about 200 cm / hr, e.g., about 10 cm / hr, about 15 cm / hr, about 20 cm / hr, about 25 cm / hr, about 30 cm / hr, about 35 cm / hr, about 40 cm / hr, about 45 cm / hr, about 50 cm / hr, about 55 cm / hr, about 60 cm / hr, about 65 cm / hr, about 70 cm / hr, about 75 cm / hr, about 80 cm / hr, about 85 cm / hr, about 90 cm / hr, about 95 cm / hr / hr, about 100 cm / hr, about 105 cm / hr, about 110 cm / hr, about 115 cm / hr, about 120 cm / hr, about 125 cm / hr, about 130 cm / hr, about 135 cm / hr, about 140 cm / hr, about 145 cm / hr, about 150 cm / hr, about 155 cm / hr, about 160 cm / hr, about 165 cm / hr, about 170 cm / hr, about 175 cm / hr, about 180 cm / hr, about 185 cm / hr, about 190 cm / hr, about 195 cm / hr, or about 200 cm / hr. Depending on the resin, the flow rate can be up to 600 cm / hr in some embodiments.

[0169] In some embodiments, the one or more buffers further comprise one or more additional substances selected from the group consisting of non-reducing sugars, sugar alcohols, and polyols. In some embodiments, the one or more buffers further comprise one or more non-reducing sugars. In some embodiments, non-reducing sugars include, but are not limited to, sucrose, trehalose, mannitol, sorbitol, galactitol, and / or xylitol. In some embodiments, the one or more buffers further comprise one or more sugar alcohols. In some embodiments, the one or more buffers further comprise one or more polyols. In some embodiments, the sugar alcohols or polyols include, but are not limited to, mannitol, xylitol, erythritol, threitol, sorbitol, and / or glycerol. In some embodiments, the buffer further comprises sorbitol, mannitol, xylitol, sucrose, trehalose, ethylene glycol, propylene glycol, glycerol, 1,2,3-propanetriol, meso-erythritol, and / or erythritol (meso-1,2,3,4-butanetriol).

[0170] In some embodiments, the buffer and chelator combination for the size exclusion chromatography method comprises citrate, malate (malic acid), or tartrate (tartaric acid).

[0171] D. Immunoaffinity purification In some embodiments, a solution containing pro-rVWF, mat-rVWF / rVWF-PP complex, mat-rVWF, and / or rVWF propeptide (rVWF-PP) is obtained by immunoaffinity purification, for example, using a monoclonal antibody column. In some embodiments, the monoclonal antibody column contains a FVIII monoclonal antibody. In some embodiments, the monoclonal antibody column contains a VWF monoclonal antibody. Such columns and methods are known and described in the art. See, for example, Zimmerman et al. (U.S. Patent No. 4,361,509; incorporated herein by reference for all purposes), which describes a method for purifying factor VIII in which the factor VIII / VWF complex is bound to a monoclonal anti-VWF antibody and factor VIII is dissociated from the complex with CaCl ions. The immunoaffinity carriers, which still have adsorbed vWF, are regenerated by a chaotropic agent, particularly NaSCN, ie, a vWF / NaSCN solution is produced as a by-product and discarded.

[0172] Other methods include those described in U.S. Patent No. 6,579,723, the entire contents of which are incorporated herein by reference, which describes a method for recovering highly purified vWF or factor VIII / vWF complexes using immunoaffinity chromatography. Such methods recover VWF from an immunoaffinity adsorbent using an eluent containing zwitterionic species. The presence of zwitterionic species allows for the use of mild conditions during preparation, facilitating retention of molecular integrity, activity, and incorporation of the recovered protein into pharmaceutical formulations without the need for additional stabilizers or preservatives. Such methods can be used in conjunction with current purification methods to obtain solutions containing pro-rVWF, mat-rVWF / rVWF-PP complexes, mat-rVWF, and / or rVWF propeptide (rVWF-PP). In some embodiments, immunoaffinity purification is optionally performed prior to step (a) in any of the purification procedures described herein, including procedures based on cation exchange, anion exchange, and / or size exclusion chromatography procedures.

[0173] E. Free mature VWF In some embodiments, the compositions provided herein have host cell (HC) impurity levels of 2.0 ppm or less, e.g., 2.0 ppm, 1.9 ppm, 1.8 ppm, 1.7 ppm, 1.6 ppm, 1.5 ppm, 1.4 ppm, 0.3 ppm, 1.2 ppm, 1.1 ppm, 1.0 ppm, 0.9 ppm, 0.8 ppm, 0.7 ppm, 0.6 ppm, 0.5 ppm, 0.4 ppm, 0.3 ppm, 0.2 ppm, 0.1 ppm, 0.09 ppm, 0.08 ppm, 0.07 ppm, 0.06 ppm, 0.05 ppm, 0.04 ppm, 0.03 ppm, 0.02 ppm, 0.01 ppm or less. In other embodiments, the compositions provided herein have host cell impurity levels of 0.6 ppm or less, e.g., 0.6 ppm, 0.5 ppm, 0.4 ppm, 0.3 ppm, 0.2 ppm, 0.1 ppm, 0.09 ppm, 0.08 ppm, 0.07 ppm, 0.06 ppm, 0.05 ppm, 0.04 ppm, 0.03 ppm, 0.02 pm, 0.01 pm or less.

[0174] In some embodiments, the compositions provided herein have a host cell (HC) impurity level of 5.0% or less (e.g., ≦5.0%). In some embodiments, the compositions provided herein have a host cell (HC) impurity level of 4.0% or less (e.g., ≦4.0%). In some embodiments, the compositions provided herein have a host cell (HC) impurity level of 3.0% or less (e.g., ≦3.0%). In some embodiments, the compositions provided herein have a host cell (HC) impurity level of 2.0% or less (e.g., ≦1.0%). In some embodiments, the compositions provided herein have a host cell (HC) impurity level of 2.0% or less (e.g., ≦1.0%). In some embodiments, the host cell (HC) impurity level is 0.9% or less (e.g., ≦0.9%). In some embodiments, the host cell (HC) impurity level is 0.8% or less (e.g., ≦0.8%). In some embodiments, the host cell (HC) impurity level is 0.7% or less (e.g., ≦0.7%). In some embodiments, the host cell (HC) impurity level is 0.6% or less (e.g., ≦0.6%). In some embodiments, the host cell (HC) impurity level is 0.5% or less (e.g., ≦0.5%). In some embodiments, the host cell (HC) impurity level is 0.4% or less (e.g., ≦0.4%). In some embodiments, the host cell (HC) impurity level is 0.3% or less (e.g., ≦0.3%). In some embodiments, the host cell (HC) impurity level is 0.2% or less (e.g., ≦0.2%). In some embodiments, the host cell (HC) impurity level is 0.1% or less (e.g., ≦0.1%).

[0175] In some embodiments, rVWF-PP impurities are less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, less than 0.1%, or less than 0.05%. In some embodiments, rVWF-PP impurities are less than 15%. In some embodiments, rVWF-PP impurities are less than 10%. In some embodiments, rVWF-PP impurities are less than 5%. In some embodiments, rVWF-PP impurities are less than 4%. In some embodiments, rVWF-PP impurities are less than 3%. In some embodiments, rVWF-PP impurities are less than 2%. In some embodiments, rVWF-PP impurities are less than 1%. In some embodiments, rVWF-PP impurities are less than 0.5%. In some embodiments, rVWF-PP impurities are less than 0.4%. In some embodiments, the rVWF-PP impurities are less than 0.3%. In some embodiments, the rVWF-PP impurities are less than 0.2%. In some embodiments, the rVWF-PP impurities are less than 0.1%. In some embodiments, the rVWF-PP impurities are less than 0.05%.

[0176] Table 1. Exemplary VWF-PP clearance capacities TIFF0007818898000001.tif51156 * : Pre-maturation before loading or complete maturation by on-column in vitro maturation (currently in process and claimed in another patent)

[0177] F. Recombinant VWF Production The free mature recombinant von Willebrand factor (rVWF) of the present invention can be produced recombinantly. Those skilled in the art will recognize useful methods for expressing recombinant proteins in host cells. In some instances, the method involves expressing a nucleic acid sequence encoding rVWF in host cells, such as CHO cells, and culturing the resulting host cells under specific conditions to produce rVWF, preproVWF, proVWF, etc.

[0178] In certain embodiments, the nucleic acid sequence containing the coding sequence for VWF can be an expression vector. The vector can be delivered by a virus or can be a plasmid. The nucleic acid sequence encoding a protein can be a specific gene or a biologically functional portion thereof. In one embodiment, the protein is at least a biologically active portion of VWF. The nucleic acid sequence can further include other sequences suitable for regulated protein expression, such as promoter sequences, enhancers, TATA boxes, transcription initiation sites, polylinkers, restriction enzyme sites, polyA sequences, protein processing sequences, and selection markers, which are generally known to those skilled in the art.

[0179] A wide variety of vectors can be used for the expression of VWF, and can be selected from eukaryotic expression vectors. Examples of vectors for eukaryotic expression include: (i) for expression in yeast, vectors such as pAO, pPIC, pYES, and pMET, which use promoters such as AOX1, GAP, GAL1, and AUG1; (ii) for expression in insect cells, vectors such as pMT, pAc5, pIB, pMIB, and pBAC, which use promoters such as PH, p10, MT, Ac5, OpIE2, gp64, and polh; and (iii) for expression in mammalian cells, vectors such as pSVL, pCMV, pRc / RSV, pcDNA3, and pBPV, as well as vectors derived from viruses such as vaccinia virus, adeno-associated virus, herpes virus, and retrovirus, which use promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV, and β-actin.

[0180] In some aspects, the rVWF used in the methods of the present invention is produced by expression in mammalian cell culture using methods known in the art. In certain embodiments, the mammalian culture comprises CHO cells. In further embodiments, the rVWF is co-expressed with recombinant factor VIII (rFVIII) in the same culture. In such embodiments, the rVWF and rFVIII are purified together (co-purified) or separately using methods known in the art. In other embodiments, the rVWF is expressed in a culture that does not contain rFVIII.

[0181] In some embodiments, rVWF is expressed in and isolated from a suitable eukaryotic host system. Examples of eukaryotic cells include, but are not limited to, mammalian cells such as CHO, COS, HEK293, BHK, SK-Hep, and HepG2; insect cells such as SF9, SF21, S2, and High Five cells; and yeast cells such as Saccharomyces or Schizosaccharomyces. In one embodiment, VWF can be expressed in yeast cells, insect cells, avian cells, mammalian cells, and the like. For example, it can be expressed in a human cell line, a hamster cell line, or a mouse cell line. In a specific embodiment, the cell line is a CHO, BHK, or HEK cell line. Generally, mammalian cells, such as CHO cells derived from a continuous cell line, can be used to express the VWF of the present invention. In a specific example, the VWF protein is expressed and isolated from a CHO cell expression system.

[0182] VWF can be produced in a cell culture system or according to any cell culture method recognized by those skilled in the art. In some embodiments, cell culture can be carried out in a large-scale bioreactor under conditions suitable for providing a high volume-specific culture surface area to achieve high cell density and protein expression. One means for providing such growth conditions is to use microcarriers for cell culture in a stirred-tank bioreactor. The concept of cell growth on microcarriers, first described by van Wezel (van Wezel, AL, Nature, 1967, 216:64-5), allows cells to adhere to the surface of small solid particles suspended in the growth medium. These methods provide a high surface-to-volume ratio and therefore efficient nutrient utilization. Furthermore, for the expression of secreted proteins in eukaryotic cell systems, an increased surface-to-volume ratio allows for higher levels of secretion and higher protein yields in the culture supernatant. Finally, these methods allow for easy scale-up of eukaryotic expression cultures.

[0183] VWF-expressing cells can be bound to spherical or porous microcarriers during cell culture growth. The microcarriers can be selected from the group of microcarriers based on dextran, collagen, plastic, gelatin, cellulose, etc., as described by Butler (1988. In: Spier & Griffiths, Animal Cell Biotechnology 3:283-303). Alternatively, cells can be grown to biomass on spherical microcarriers and subcultured when they reach the final fermenter biomass and before the expressed protein is produced on porous microcarriers, or vice versa. Suitable spherical microcarriers can include smooth microcarriers such as Cytodex™ 1, Cytodex™ 2, and Cytodex™ 3 (GE Healthcare), as well as porous microcarriers such as Cytopore™ 1, Cytopore™ 2, Cytoline™ 1, and Cytoline™ 2 (GE Healthcare).

[0184] In further embodiments, pro-VWF is exposed to furin in vitro to cleave the VWF propeptide from immature VWF. In some embodiments, the furin used for propeptide cleavage is recombinant furin.

[0185] In certain embodiments, rVWF is expressed in cells cultured in a cell culture medium that produces high molecular weight rVWF. The terms "cell culture medium," "cell culture medium or medium," and "cell culture supernatant" refer to aspects of cell culture processes generally known in the art. In the context of the present invention, cell culture medium can include cell culture medium and cell culture supernatant. Cell culture medium, optionally with supplements, is exogenously added to the cell culture medium to provide nutrients and other components for culturing VWF-expressing cells. Cell culture supernatant refers to a cell culture medium that contains nutrients and other components from the cell culture medium as well as products released, metabolized, and / or excreted by the cells during culture. In further embodiments, the medium is animal protein-free and can be chemically defined. Methods for preparing animal protein-free and chemically defined culture media are known in the art, e.g., US2006 / 0094104, US2007 / 0212770, and US2008 / 0009040, which are incorporated herein by reference for all purposes, particularly for all teachings related to cell culture media. "Protein-free" and related terms refer to proteins that are exogenous to the culture or derived from sources other than the cells, and which are naturally removed during growth. In another embodiment, the culture medium is polypeptide-free. In another embodiment, the culture medium is serum-free. In another embodiment, the culture medium is animal protein-free. In another embodiment, the culture medium is animal component-free. In another embodiment, the culture medium contains a protein, e.g., a serum-derived animal protein, such as fetal bovine serum. In another embodiment, the culture contains an exogenously added recombinant protein. In another embodiment, the protein is derived from a certified pathogen-free animal. As used herein, the term "chemically defined" means that the medium does not contain undefined supplements, such as extracts of animal components, organs, glands, plants, or yeast. Thus, each component of a chemically defined medium is precisely defined. In a preferred embodiment, the medium is both animal component-free and protein-free.

[0186] In certain embodiments, the culture of VWF-expressing cells can be maintained for at least about 7 days, or at least about 14 days, 21 days, 28 days, or at least about 5 weeks, 6 weeks, 7 weeks, or at least about 2 months, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 months or more. The cell density at which the cell culture is maintained for the production of recombinant VWF protein depends on the culture conditions and medium used for protein expression. One skilled in the art can readily determine the optimal cell density for a cell culture producing VWF. In one embodiment, the culture is maintained at a cell density of about 0.5x10 for an extended period of time. 6 ~4x10 7 In another embodiment, the cell density is maintained at about 1.0x10 cells / ml for an extended period of time. 6 ~about 1.0x10 7 In another embodiment, the cell density is maintained at about 1.0x10 cells / ml for an extended period of time. 6 ~about 4.0x10 6 In another embodiment, the cell density is maintained at about 1.0x10 cells / ml for an extended period of time. 6 ~about 4.0x10 6 In yet another embodiment, the cell density is maintained at about 2.0 x 10 cells / ml. 6 ~about 4.0x10 6 , or approximately 1.0x10 6 ~about 2.5x10 6 , or about 1.5x10 6 ~about 3.5x10 6 or any other similar range of concentration for an extended period of time. After a suitable period in cell culture, the rVWF can be isolated from the expression system using methods known in the art.

[0187] In certain embodiments, the cell density of the continuous cell culture for rVWF production is maintained at 2.5x10 for extended periods. 6 In another particular embodiment, the cell density is maintained at a concentration of 2.0x10 cells / mL or less. 6 cells / mL, 1.5x10 6 cells / mL, 1.0x10 6cells / mL, 0.5x10 6 In one embodiment, the cell density is maintained at 1.5x10 cells / mL or less. 6 cells / mL~2.5x10 6 Maintain cells / mL.

[0188] In one embodiment of the above cell culture, the cell culture medium comprises a medium supplement containing copper. Such cell culture medium is described, for example, in U.S. Patent No. 8,852,888 and U.S. Patent No. 9,409,971, which are incorporated herein by reference in their entirety for all purposes, particularly for all teachings related to cell culture methods and compositions for producing recombinant VWF.

[0189] The polynucleotide and amino acid sequences of prepro-VWF are set forth in SEQ ID NO:1 and SEQ ID NO:2, respectively, and are available under GenBank accession numbers NM_000552 (Homo sapiens von Willebrand factor (VWF) mRNA) and NP_000543, respectively. The amino acid sequence corresponding to the mature VWF protein is set forth in SEQ ID NO:3 (corresponding to amino acids 764 to 2813 of the full-length prepro-VWF amino acid sequence). In some embodiments, the VWF exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to the sequence of SEQ ID NO:3. In some embodiments, the mat-rVWF of the present invention exhibits at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% identity to the sequence of SEQ ID NO: 3. See, e.g., U.S. Patent No. 8,597,910, U.S. Patent Publication No. 2016 / 0129090, and Figure 60.

[0190] A useful form of rVWF has at least the properties of in vivo stabilization, e.g., binding of at least one factor VIII (FVIII) molecule and, optionally, a pharmacologically acceptable glycosylation pattern. Specific examples include VWF lacking the A2 domain and therefore resistant to proteolysis (Lankhof et al., Thromb. Haemost. 77:1008-1013, 1997), and the VWF fragment from Val449 to Asn730, which contains the glycoprotein 1b-binding domain and collagen and heparin-binding sites (Pietu et al., Biochem. Biophys. Res. Commun. 164:1339-1347, 1989). In one embodiment, determining the ability of VWF to stabilize at least one FVIII molecule is performed in a VWF-deficient mammal according to methods known in the art.

[0191] The rVWF of the present invention can be produced by any method known in the art. Specific examples are disclosed in WO 86 / 06096, published October 23, 1986, and U.S. Patent Application No. 07 / 559,509, filed July 23, 1990, which are incorporated herein by reference for methods of producing recombinant VWF. Thus, methods are known in the art for (i) producing recombinant DNA by genetic modification, e.g., by RNA reverse transcription and / or DNA amplification, (ii) introducing the recombinant DNA into prokaryotic or eukaryotic cells, e.g., by transfection using electroporation or microinjection, (iii) culturing the transformed cells, e.g., in a continuous or batch mode, (iv) expressing VWF, e.g., constitutively or upon induction, and (v) isolating VWF, e.g., from the culture medium or by recovering the transformed cells, and (vi) obtaining purified rVWF, e.g., via anion exchange chromatography or affinity chromatography. In one embodiment, recombinant VWF is produced in transformed host cells using recombinant DNA techniques well known in the art. For example, the coding sequence for the polypeptide can be excised from the DNA using appropriate restriction enzymes. Alternatively, in another embodiment, the DNA molecule is synthesized using chemical synthesis techniques such as the phosphoramidate method. In yet another embodiment, a combination of these techniques is used.

[0192] The present invention also provides a vector encoding a polypeptide of the present invention in a suitable host. The vector comprises a polynucleotide encoding the polypeptide operably linked to a suitable expression control sequence. Methods for achieving this operably linkage, either before or after inserting the polynucleotide into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosome binding sites, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved in the control of transcription or translation. The resulting vector, carrying the polynucleotide therein, is used to transform a suitable host. This transformation may be carried out using methods well known in the art.

[0193] Any of a large number of available, well-known host cells may be used in the practice of the present invention. The selection of a particular host depends on many factors recognized in the art, including, for example, compatibility with the selected expression vector, toxicity of the peptide encoded by the DNA molecule, transformation rate, ease of peptide recovery, expression characteristics, biosafety, and cost. A balance of these factors must be determined, with the understanding that not all host cells are equally effective in expressing a particular DNA sequence. Within these general guidelines, useful microbial host cells include, but are not limited to, bacteria, yeast and other fungi, insect, plant, mammalian (including human) cells in culture, or other hosts known in the art.

[0194] The transformed host cells are cultured under conventional fermentation conditions to express the desired compound. Such fermentation conditions are well known in the art. Finally, the polypeptide is purified from the culture medium or the host cells themselves by methods well known in the art.

[0195] Depending on the host cell used to express the compounds of the present invention, carbohydrate (oligosaccharide) groups are optionally attached to sites known to be glycosylation sites in proteins. Generally, O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues, and N-linked oligosaccharides are attached to asparagine (Asn) residues when they are part of the sequence Asn-X-Ser / Thr, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids, not counting proline. The structures of N-linked and O-linked oligosaccharides, and the sugar residues found in each type, differ. One type of sugar commonly found in both N-linked and O-linked oligosaccharides is N-acetylneuraminic acid (called sialic acid). Sialic acid is usually the terminal residue of both N-linked and O-linked oligosaccharides, and its negative charge, in one aspect, confers acidic properties to glycosylated compounds. Such site(s) may be incorporated into the linker of the compounds of the invention and are preferably glycosylated by the cell during recombinant production of the polypeptide compound (e.g., in mammalian cells such as CHO, BHK, COS, etc.) In other embodiments, such sites are glycosylated by synthetic or semi-synthetic procedures known in the art.

[0196] In some embodiments, sialylation (also called sialylation) can be performed on a column as part of the purification procedures described herein (including anion exchange, cation exchange, size exclusion, and / or immunoaffinity methods). In some embodiments, sialylation increases the stability of rVWF compared to non-sialylated rVWF. In some embodiments, sialylation increases the stability of rVWF in the circulation (e.g., after administration to a subject) compared to non-sialylated rVWF. In some embodiments, the increased stability of salivary rVWF results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more increase compared to non-sialylated rVWF. In some embodiments, sialylation increases the half-life of rVWF compared to non-sialylated rVWF. In some embodiments, sialylation increases the half-life of rVWF in the circulation (e.g., after administration to a subject) compared to non-sialylated rVWF. In some embodiments, the increased half-life of sialylated rVWF results in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more increase compared to non-sialylated rVWF. In some embodiments, the increased half-life of sialylated rVWF results in rVWF that is stable in the circulation for 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours, 24 hours or more (e.g., after administration to a subject) compared to non-sialylated rVWF. In some embodiments, sialylation increases the number of 2,3 sialylation sites and / or 2,6 sialylation sites. In some embodiments, sialylation is increased by adding a 2,3 sialyltransferase and / or a 2,6 sialyltransferase and CMP-NANA (cytidine-5'-monophospho-N-acetylneuraminic acid sodium salt) as an additional buffering step. In some embodiments, sialylation is increased by adding a 2,3 sialyltransferase and CMP-NANA (cytidine-5'-monophospho-N-acetylneuraminic acid sodium salt) as an additional buffering step.In some embodiments, 2,3 sialylation is increased by adding a 2,3 sialyltransferase and CMP-NANA (cytidine-5'-monophospho-N-acetylneuraminic acid sodium salt) as an additional buffering step. In some embodiments, to increase sialylation, the bound protein (e.g., bound rVWF) is treated with a sialidase (e.g., neuraminidase) to remove 2,3 sialylation, followed by a washing step to remove the sialidase and introduce 2,6 sialylation. In some embodiments, 2,6 sialylation is introduced by adding a 2,6 sialyltransferase and CMP-NANA.

[0197] In some embodiments, 2,6 sialylation is increased by adding a 2,6 sialyltransferase and CMP-NANA (cytidine-5'-monophospho-N-acetylneuraminic acid sodium salt) as an additional buffering step. In some embodiments, 2,3 sialylation and / or 2,6 sialylation is increased by adding a 2,3 sialyltransferase and / or a 2,6 sialyltransferase and CMP-NANA (cytidine-5'-monophospho-N-acetylneuraminic acid sodium salt) as an additional buffering step. In some embodiments, CMP-NANA is chemically or enzymatically modified to transfer the modified sialic acid to a neutral site. In some embodiments, sialylation is carried out by loading rVWF onto a resin, washing with one or more buffers described herein to deplete undesired impurities, adding one or more buffers containing a sialyltransferase and CMP-NANA under conditions that allow further sialylation, washing with one or more buffers to deplete excess sialylation reagent, and eluting the enhanced rVWF (e.g., rVWF with increased sialylation) with one or more buffers. In some embodiments, the sialylation process is carried out as part of a cation exchange, anion exchange, size exclusion, or immunoaffinity purification method, as described herein.

[0198] Alternatively, the compound can be prepared by synthesis, for example, using solid phase synthesis techniques. Suitable techniques are well known in the art and include those described in Merrifield (1973), Chem. Polypeptides, pp. 335-61 (Katsoyannis and Panayotis eds.); Merrifield (1963), J. Am. Chem. Soc. 85: 2149; Davis et al. (1985), Biochem. Intl. 10: 394-414; Stewart and Young (1969), Solid Phase Peptide Synthesis; U.S. Patent No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105-253; and Erickson et al. (1976), The Proteins (3rd ed.) 2: 257-527'. Solid phase synthesis is the preferred technique for producing individual peptides because it is the most cost-effective method for producing small peptides.

[0199] Fragments, variants, and analogs of VWF can be produced according to methods well known in the art. Polypeptide fragments can be prepared using, but not limited to, enzymatic cleavage (e.g., trypsin, chymotrypsin) and recombinant means to generate polypeptide fragments having specific amino acid sequences. Polypeptide fragments can also be generated that contain regions of the protein with specific activities, such as the multimerization domain or any other identifiable VWF domain known in the art.

[0200] Methods for producing polypeptide analogs are also well known. Amino acid sequence analogs of polypeptides can be substitution, insertion, addition, or deletion analogs. Deletion analogs, including polypeptide fragments, lack one or more residues of the native protein that are not essential for function or immunogenic activity. Insertion analogs, for example, have the addition of an amino acid(s) at a non-terminal site in the polypeptide. This analog can include, for example, but is not limited to, the insertion of an immunoreactive epitope or simply a single residue. Addition analogs, including polypeptide fragments, have the addition of one or more amino acids at one or both ends of the protein, including, for example, fusion proteins. Combinations of the above analogs are also contemplated.

[0201] Substitution analogs generally involve replacing one wild-type amino acid with another at one or more sites within a protein, and may be designed to adjust one or more properties of a polypeptide without completely eliminating other functions or properties. In one embodiment, the substitution is a conservative substitution. A "conservative amino acid substitution" is a substitution of one amino acid with an amino acid having a side chain or similar chemical characteristics. Similar amino acids for conservative substitution include those with acidic side chains (glutamic acid, aspartic acid); basic side chains (arginine, lysine, histidine); polar amide side chains (glutamine, asparagine); hydrophobic aliphatic side chains (leucine, isoleucine, valine, alanine, glycine); aromatic side chains (phenylalanine, tryptophan, tyrosine); small side chains (glycine, alanine, serine, threonine, methionine); or aliphatic hydroxyl side chains (serine, threonine).

[0202] In one embodiment, analogs are substantially homologous or substantially identical to the recombinant VWF from which they are derived. Analogs include those that retain at least some biological activity of the wild-type polypeptide, such as blood clotting activity.

[0203] Contemplated polypeptide variants include polypeptides that have been chemically modified by techniques such as, but not limited to, ubiquitination, glycosylation, including polysialylation (or polysialylation), conjugation with a therapeutic or diagnostic agent, labeling, covalent polymer attachment such as pegylation (derivatization with polyethylene glycol), introduction of a non-hydrolyzable bond, and chemically synthesized insertion or substitution of an amino acid, such as ornithine, that does not normally occur in human proteins. Variants retain the same or essentially the same binding properties as the unmodified molecules of the invention. Such chemical modifications may involve direct or indirect (e.g., via a linker) attachment of an agent to the VWF polypeptide. In the case of indirect attachment, the linker may be hydrolyzable or non-hydrolyzable.

[0204] In one aspect, the preparation of a PEGylated polypeptide analog involves (a) reacting a polypeptide with polyethylene glycol (e.g., a reactive ester or aldehyde derivative of PEG) under conditions such that the polypeptide of the conjugated construct is conjugated to one or more PEG groups, and (b) obtaining the reaction product(s). Generally, optimal reaction conditions for an acylation reaction are determined based on known parameters and the desired results. For example, the higher the PEG:protein ratio, the greater the proportion of polyPEGylated product. In some embodiments, the conjugated construct has a single PEG moiety at the N-terminus. Polyethylene glycol (PEG) can be attached to a blood clotting factor to, for example, provide a longer half-life in vivo. The PEG group can be of any convenient molecular weight and can be linear or branched. The average molecular weight of the PEG ranges from about 2 kilodaltons ("kD") to about 100 kDa, about 5 kDa to about 50 kDa, or about 5 kDa to about 10 kDa. In certain embodiments, the PEG group is attached to the blood clotting factor through a native or modified reactive group (e.g., an aldehyde, amino, thiol, or ester group) on the PEG moiety, by acylation or reductive alkylation to a reactive group (e.g., an aldehyde, amino, or ester group) on the blood clotting factor, or by any other technique known in the art.

[0205] Methods for preparing polysialylated polypeptides are described in U.S. Patent Publication 20060160948, Fernandes et Gregoriadis; Biochim. Biophys. Acta 1341:26-34, 1997, and Saenko et al., Haemophilia 12:42-51, 2006. Briefly, a solution of colominic acid (CA) containing 0.1 M NaIO4 is stirred in the dark at room temperature to oxidize the CA. The activated CA solution is dialyzed against, for example, 0.05 M sodium phosphate buffer, pH 7.2, in the dark, and this solution is added to the rVWF solution and incubated at room temperature for 18 hours under gentle stirring in the dark. Optionally, the free reagent is separated from the rVWF-polysialic acid complex by, for example, ultrafiltration / diafiltration. Conjugation of rVWF to polysialic acid is achieved using glutaraldehyde as a cross-linking reagent (Migneault et al., Biotechniques 37:790-796, 2004).

[0206] In another aspect, it is further contemplated that the polypeptides of the present invention are fusion proteins with a second agent that is a polypeptide. In one embodiment, the second agent that is a polypeptide is, but is not limited to, an enzyme, a growth factor, an antibody, a cytokine, a chemokine, a cell surface receptor, an extracellular domain of a cell surface receptor, a cell adhesion molecule, or a fragment or active domain of the above proteins. In a related embodiment, the second agent is a blood clotting factor such as factor VIII, factor VII, and / or factor IX. In some embodiments, the second agent is a fusion protein. Contemplated fusion proteins are produced by chemical or recombinant techniques well known in the art. In some embodiments, the fusion protein is a rVWF-FVIII fusion protein. In some embodiments, the fusion protein is a rVWF-FVIII fusion protein, in which active FVIII is embedded in a VWF motif. In some embodiments, the fusion protein is a rVWF-FVIII fusion protein, in which active FVIII is embedded in a VWF motif such that VWF is full-length. In some embodiments, the fusion protein is a rVWF-FVIII fusion protein, in which active FVIII is embedded in a VWF motif and a portion of the VWF sequence is deleted and replaced with FVIII sequence. In some embodiments of the rVWF-FVIII fusion protein, the FVIII is a B-domain deleted FVIII. In some embodiments of the rVWF-FVIII fusion protein, the FVIII-B domain is replaced with an N-glycosylation-rich domain. In some embodiments of the rVWF-FVIII fusion protein, the vWF-N-glycosylation-rich domain is fused to full-length FVIII and / or its truncated forms.

[0207] In some embodiments of the rVWF-FVIII fusion protein, the fusion protein comprises: a VWF peptide comprising positions 764 to 1336 of the VWF peptide; a FVIII peptide comprising positions 24 to 760 of the FVIII heavy chain peptide; a VWF peptide comprising positions 2218 to 2593 of the VWF peptide; a FVIII peptide comprising positions 1333 to 2351 of the FVIII light chain peptide; and A VWF peptide comprising positions 2620 to 2813 of the VWF peptide. In this embodiment of the rVWF-FVIII fusion protein, the amino acid positions are counted from the first position, which includes Pro and / or the signal peptide. In this embodiment of the rVWF-FVIII fusion protein, position 764 in VWF corresponds to position 1 of mature rVWF (mat-rVWF), and position 20 in FVIII corresponds to position 1 of the mature FVIII peptide. In some embodiments of the rVWF-FVIII fusion protein, the sequence of the fusion protein is shown in Figure 64.

[0208] In some embodiments of the rVWF-FVIII fusion protein, the fusion protein comprises: a FVIII peptide comprising positions 19 to 760 of the FVIII heavy chain of the FVIII heavy chain peptide; A VWF peptide comprising positions 2218 to 2593 of the VWF peptide, and A FVIII peptide comprising positions 1333 to 2351 of the FVIII light chain peptide. In this embodiment of the rVWF-FVIII fusion protein, the amino acid positions are counted from the first position, which includes Pro and / or the signal peptide. In this embodiment of the rVWF-FVIII fusion protein, position 764 in VWF corresponds to position 1 of mature rVWF (mat-rVWF), and position 20 in FVIII corresponds to position 1 of the mature FVIII peptide. In some embodiments of the rVWF-FVIII fusion protein, the sequence of the fusion protein is shown in Figure 65.

[0209] In another embodiment, it is also contemplated that prepro-VWF and pro-VWF polypeptides provide therapeutic benefit in the formulations of the invention. For example, U.S. Patent No. 7,005,502 describes pharmaceutical formulations containing amounts of pro-VWF sufficient to induce thrombin generation in vitro. In addition to recombinant, biologically active fragments, variants, or other analogs of native mature VWF, the present invention contemplates the use of recombinant, biologically active fragments, variants, or analogs of prepro-VWF (as set forth in SEQ ID NO:2) or pro-VWF polypeptides (amino acid residues 23-764 of SEQ ID NO:2) in the formulations described herein.

[0210] Polynucleotides encoding fragments, variants, and analogs can be readily generated by artisans to encode biologically active fragments, variants, or analogs of naturally occurring molecules that have the same or similar biological activity as the naturally occurring molecules. In various embodiments, these polynucleotides are prepared using PCR techniques, digestion / ligation of DNA-encoding molecules, and the like. Thus, one skilled in the art can generate single-base changes in DNA strands, resulting in codon changes and missense mutations, using any method known in the art, including, but not limited to, site-directed mutagenesis. As used herein, the phrase "moderately stringent hybridization conditions" refers, for example, to hybridization in 50% formamide at 42°C and washing in 0.1x SSC, 0.1% SDS at 60°C. Those skilled in the art will appreciate that these conditions will vary based on the length and GC nucleotide base content of the hybridizing sequences. Standard formulas in the art are suitable for determining precise hybridization conditions. See Sambrook et al., 9.47-9.51 in Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

[0211] G. Viral Inactivation In some embodiments, the methods described herein further comprise a viral inactivation step. The viral inactivation step can be performed before, after, or simultaneously with the washing and / or elution steps, but before the recovery step. The viral inactivation treatment can inactivate lipid-enveloped viruses. In some embodiments, the viral inactivation treatment is a solvent and detergent (S / D) treatment. In some embodiments, the viral inactivation treatment comprises the use of ethylene glycol, propylene glycol in ethanol, and / or one or more organic solvent(s).

[0212] As used herein, the term "viral inactivation" or "viral inactivation" refers to a process in which a virus can no longer infect, replicate, and propagate in cells, essentially eliminating the virus. Thus, the term "viral inactivation" generally refers to a process for making the fluids disclosed herein completely free of infectious viral contaminants. Any degree of viral inactivation using the methods disclosed herein is desirable. However, it is desirable to achieve the degree of viral inactivation necessary to meet strict safety guidelines for pharmaceutical products. These guidelines are established by the WHO and are well known to those skilled in the art.

[0213] The method disclosed herein may further comprise a step of removing virus from the mixture after incubation. As used herein, the term "removing virus" or "virus removal" refers to a process of depleting virus from the mixture disclosed herein, thereby effectively extracting virus particles from the mixture. The virus may be live or inactivated. Removal is generally achieved by size exclusion chromatography or positive adsorption chromatography, in which the protein of interest binds to a chromatography resin, including, for example, an anion exchange resin or cation exchange resin described herein. After removal, the remaining amount of virus is such that it does not have a substantial long-term or permanent adverse effect when administered to a subject in need thereof, including, for example, a human.

[0214] In one embodiment, the mixture after virus removal is essentially virus-free. As used herein, the term "essentially virus-free" means that only trace amounts of virus can be detected or confirmed by the equipment or process used to detect or confirm the presence or activity of the virus, and such trace amounts of virus are insufficient to cause harm to human health. In one aspect of this embodiment, the mixture after virus removal is completely virus-free. As used herein, the term "completely virus-free" means that the presence of virus cannot be detected or confirmed within the detection range of the equipment or process used to detect or confirm the presence or activity of the virus. Because the virus is insufficient to cause harm to human health, proteins contained in the essentially virus-free or completely virus-free mixture can be used to make pharmaceutical compositions that can be safely administered to humans.

[0215] In other aspects of this embodiment, the post-virus removal mixture contains less than 10 PFU / mL of virus, e.g., less than 1 PFU / mL of virus, e.g., less than 1 x 10 -1 Viruses less than 1 × 10 PFU / mL -2 PFU / mL of virus, or 1 × 10 -3 containing PFU / mL of virus.

[0216] In still other aspects of this embodiment, the mixture after virus removal comprises less than an ID50 of viruses, e.g., at most 1 / 10 less than an ID50 of viruses, at most 1 / 100 less than an ID50 of viruses, at most 1 / 200 less than an ID50 of viruses, at most 1 / 300 less than an ID50 of viruses, at most 1 / 400 less than an ID50 of viruses, at most 1 / 500 less than an ID50 of viruses, at most 1 / 600 less than an ID50 of viruses, at most 1 / 700 less than an ID50 of viruses, at most 1 / 800 less than an ID50 of viruses, at most 1 / 900 less than an ID50 of viruses, or at most 1 / 1000 less than an ID50 of viruses.

[0217] Viral inactivation may or may not be performed in conjunction with protein purification. In some embodiments, the method includes immobilizing a protein on a support and treating the immobilized protein with a detergent-solvent mixture comprising a non-ionic detergent and an organic solvent. In some embodiments, the support is a chromatography resin. In certain embodiments, the detergent-solvent mixture comprises 1% Triton X-100, 0.3% tri-N-butyl phosphate, and 0.3% polysorbate 80 (Tween 80). The solvent-detergent mixture treatment can be continued for an extended period of time, e.g., 30 minutes to 1 hour, as long as the protein remains immobilized on the chromatography resin, e.g., a cation exchange resin; and / or the solvent-detergent treatment can be performed at 2°C to 10°C. This approach to viral inactivation surprisingly reduces the formation of protein aggregates during treatment with the detergent-solvent mixture by a significant amount, e.g., more than 50%, compared to treatment with the solvent-detergent mixture when the protein is not immobilized in solution.

[0218] In some embodiments, a method for inactivating a lipid-coated virus includes the steps of: i) providing a fluid containing an active protein; ii) mixing an organic solvent and a surfactant with the fluid, thereby creating a mixture; and iii) incubating the mixture for no more than about 120 minutes; wherein both steps (ii) and (iii) are performed at a temperature of about 20°C or less; the mixture after incubation is essentially free of viable lipid-coated virus; and wherein the protein after incubation has at least 25% of the activity provided in step (i).

[0219] In other embodiments, a protein that is essentially free of lipid-coated viruses can be obtained from a method comprising the following steps: i) providing a fluid containing an active protein; ii) mixing an organic solvent and a surfactant with the fluid, thereby creating a mixture; iii) incubating the mixture for no more than about 120 minutes; wherein both steps (ii) and (iii) are performed at a temperature of about 20°C or less; the mixture after incubation is essentially free of viable lipid-coated viruses; and wherein the protein after incubation has at least 25% of the activity of the protein provided in step (a).

[0220] In another embodiment, a method for inactivating a lipid-coated virus includes the following steps: i) providing a fluid containing an active blood coagulation protein (e.g., VWF); ii) mixing an organic solvent and a surfactant with the fluid, thereby forming a mixture; iii) incubating the mixture for no more than about 120 minutes; wherein both steps (ii) and (iii) are performed at a temperature of about 20°C or less; the mixture after incubation is essentially free of viable lipid-coated virus; and wherein the Factor VIII after incubation has at least 25% of the activity provided in step (i).

[0221] In some cases, the organic solvent is an ether, an alcohol, a dialkyl phosphate, or a trialkyl phosphate. In certain embodiments, the ether is selected from dimethyl ether, diethyl ether, ethyl propyl ether, methyl butyl ether, methyl isopropyl ether, and / or methyl isobutyl ether.

[0222] In some embodiments, the alcohol is selected from methanol, ethanol, propanol, isopropanol, n-butanol, isobutanol, n-pentanol, and / or isopentanol. In some embodiments, the dialkyl phosphate is selected from di-(n-butyl) phosphate, di-(t-butyl) phosphate, di-(n-hexyl) phosphate, di-(2-ethylhexyl) phosphate, di-(n-decyl) phosphate, and / or ethyl di(n-butyl) phosphate. In some embodiments, the trialkyl phosphate is selected from tri-(n-butyl) phosphate, tri-(t-butyl) phosphate, tri-(n-hexyl) phosphate, tri-(2-ethylhexyl) phosphate, and / or tri-(n-decyl) phosphate.

[0223] In some cases, the final concentration of the organic solvent is about 0.1% (v / v) to about 5.0% (v / v), about 0.1% (v / v) to about 1.0% (v / v), about 0.2% (v / v) to about 0.5% (v / v), or about 0.2% (v / v) to about 0.4% (v / v), or about 0.3% (v / v).

[0224] In some cases, the surfactant is selected from an ionic surfactant, a zwitterionic (amphoteric) surfactant, and / or a nonionic surfactant. The ionic surfactant may be an anionic surfactant or a cationic surfactant.

[0225] In certain embodiments, the anionic surfactant is selected from alkyl sulfates, alkyl ether sulfates, docusate, sulfonic acid fluorosurfactants, alkyl benzene sulfonates, alkyl aryl ether phosphates, alkyl ether phosphates, alkyl carboxylates, sodium lauroyl sarcosinate, and / or carboxylic acid fluorosurfactants. In some embodiments, the alkyl sulfate is selected from ammonium lauryl sulfate or sodium lauryl sulfate (SDS). In other embodiments, the alkyl ether sulfate is sodium laureth sulfate and / or sodium myreth sulfate. In some embodiments, the docusate is dioctyl sodium sulfosuccinate.

[0226] In some embodiments, the sulfonic acid fluorosurfactant is selected from perfluorooctane sulfonate (PFOS) and / or perfluorobutane sulfonate. In some embodiments, the alkyl carboxylate is selected from fatty acid salts and / or sodium stearate. In some embodiments, the carboxylic acid fluorosurfactant is perfluorononanoate and perfluorooctanoate. In some embodiments, the cationic surfactant is selected from alkyltrimethylammonium salts, cetylpyridinium chloride (CPC), polyethoxylated tallow amine (POEA), benzalkonium chloride (BAC), benzethonium chloride (BZT), 5-bromo-5-nitro-1,3-dioxane, dimethyldioctadecylammonium chloride, dioctadecyldimethylammonium bromide (DODAB), pH-dependent primary amines, pH-dependent secondary amines, and / or pH-dependent tertiary amines. In some embodiments, the alkyltrimethylammonium salt is selected from cetyltrimethylammonium bromide (CTAB) and / or cetyltrimethylammonium chloride (CTAC). In some embodiments, primary amines become positively charged at pH<10 or secondary amines become charged at pH<4.

[0227] In some embodiments, the cationic surfactant is octenidine dihydrochloride.

[0228] In some embodiments, the zwitterionic surfactant is selected from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), a sultaine, a betaine, and / or a lecithin. In some embodiments, the sultaine is cocamidopropyl hydroxysultaine. In some embodiments, the betaine is cocamidopropyl betaine.

[0229] In some embodiments, the nonionic surfactant is selected from polyoxyethylene glycol sorbitan alkyl esters, poloxamers, alkylphenol polyglycol ethers, polyethylene glycol alkylaryl ethers, polyoxyethylene glycol alkyl ethers, 2-dodecoxyethanol (LUBROL®-PX), polyoxyethylene glycol octylphenol ethers, polyoxyethylene glycol alkylphenol ethers, phenoxypolyethoxylethanol, glucoside alkyl ethers, maltoside alkyl ethers, thioglucoside alkyl ethers, digitonin, glycerol alkyl esters, alkylaryl polyether sulfates, alcohol sulfonates, sorbitan alkyl esters, cocamidoethanolamine, sucrose monolaurate, dodecyldimethylamine oxide, and / or sodium cholate. In some embodiments, the polyoxyethylene glycol sorbitan alkyl ester is selected from polysorbate 20 sorbitan monooleate (TWEEN® 20), polysorbate 40 sorbitan monooleate (TWEEN® 40), polysorbate 60 sorbitan monooleate (TWEEN® 60), polysorbate 61 sorbitan monooleate (TWEEN® 61), polysorbate 65 sorbitan monooleate (TWEEN® 65), polysorbate 80 sorbitan monooleate (TWEEN® 80), and / or polysorbate 81 sorbitan monooleate (TWEEN® 81).

[0230] In some embodiments, the poloxamer is selected from poloxamer 124 (PLURONIC® L44), poloxamer 181 (PLURONIC® L61), poloxamer 182 (PLURONIC® L62), poloxamer 184 (PLURONIC® L64), poloxamer 188 (PLURONIC® F68), poloxamer 237 (PLURONIC® F87), poloxamer 338 (PLURONIC® L108), and / or poloxamer 407 (PLURONIC® F127).

[0231] In some embodiments, the polyoxyethylene glycol alkyl ether is selected from octaethylene glycol monododecyl ether, pentaethylene glycol monododecyl ether, BRIJ® 30, and / or BRIJ® 35.

[0232] In some cases, the polyoxyethylene glycol octylphenol ether is selected from polyoxyethylene (4-5) pt-octylphenol (TRITON® X-45) and / or polyoxyethylene octylphenyl ether (TRITON® X-100). In some embodiments, the polyoxyethylene glycol alkylphenol ether is nonoxynol-9.

[0233] In some embodiments, the phenoxypolyethoxylethanol is selected from nonylphenoxypolyethoxylethanol and / or octylphenoxypolyethoxylethanol. In some embodiments, the glucoside alkyl ether is octyl glucopyranoside. In some embodiments, the maltoside alkyl ether is dodecyl maltopyranoside. In some embodiments, the thioglucoside alkyl ether is heptyl thioglucopyranoside. In some embodiments, the glycerol alkyl ester is glyceryl laurate. In some embodiments, the cocamide ethanolamine is selected from cocamide monoethanolamine and / or cocamide diethanolamine.

[0234] In some embodiments, the final concentration of the surfactant is about 0.1% (v / v) to about 10.0% (v / v), or about 0.5% (v / v) to about 5.0% (v / v). In some cases, the surfactant is a plurality of surfactants.

[0235] Useful methods for viral inactivation are described, for example, in U.S. Pat. Nos. 6,190,609 and 9,315,560, and U.S. Patent Publication No. 2017 / 0327559, the disclosures of which are incorporated herein by reference in their entireties.

[0236] Viral inactivation can be carried out as recognized by those skilled in the art. For example, detergents such as, but not limited to, the solvent tri(n-butyl)phosphate (TNBP) and polysorbate 80 and Triton X-100 are effective in inactivating lipid-enveloped viruses. Viral inactivation can be carried out at room temperature, such as 14°C to about 25°C, for about 1 hour or more. In some cases, the incubation time is 2 hours or less.

[0237] In some embodiments, the virus inactivation treatment is stopped by adding a buffer containing a sodium citrate buffer to the virus inactivation material. In some cases, the sodium citrate buffer comprises about 40 mM to about 100 mM sodium citrate buffer, for example, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM sodium citrate buffer.

[0238] H.VWF mature Furin is part of a protein family called SPC (subtilisin-like proprotein convertase), PC (proprotein convertase), or in some cases PACE (paired basic amino acid cleaving enzyme). Members of the furin protein family include, but are not limited to, furin, Kex2, PC2, PC1 / PC3, PACE4, PC4, PC5, and / or PC7. As part of the present invention, methods are provided for maturing pro-VWF (pro-rVWF) into mat-VWF / VWF-PP (mat-rVWF / rVWF-PP) complexes by treatment with furin. Any of these furin family members can be used in methods of VWF maturation.

[0239] In some embodiments, pro-VWF is furin-matured on an anion exchange column or resin, a cation exchange column or resin, or as part of a size-exclusion chromatography procedure. In some embodiments, pro-VWF is furin-matured on an anion exchange column or resin and / or as part of an anion exchange chromatography procedure. In some embodiments, pro-VWF is furin-matured on a cation exchange column or resin and / or as part of a cation exchange chromatography procedure. In some embodiments, pro-VWF is furin-matured as part of a size-exclusion chromatography procedure. Such methods are described, for example, in U.S. Pat. No. 8,058,411, which is incorporated herein by reference in its entirety for all purposes.

[0240] To facilitate the maturation process and provide pro-VWF immobilized on the resin at a high concentration, in some embodiments of the present invention, the chromatography resin is packed into a chromatography column. Packing the chromatography resin into a column is advantageous because the concentration of pro-VWF during in vitro maturation affects maturation efficiency. Furthermore, the use of a chromatography column allows for efficient control of maturation parameters in a more reproducible manner, making in vitro VWF maturation easier to perform. In some embodiments, the furin concentration is about 1, about 2, about 3, or about 4 units of recombinant active furin per IU of VWF:Ag (10 μg of pro-rVWF). In some embodiments, the furin concentration is about 2-3 units of recombinant active furin per IU of VWF:Ag (10 μg of pro-rVWF). In some embodiments, the furin concentration is about 1-2 units of recombinant active furin per IU of VWF:Ag (10 μg of pro-rVWF). In some embodiments, the furin concentration is about 2 units of recombinant active furin per IU of VWF:Ag (10 μg of pro-rVWF).

[0241] In some embodiments, when pro-VWF is immobilized on an anion exchange resin and incubated with a solution that exhibits pro-VWF convertase activity, the conductivity measured at 25°C is less than 25 mS / cm. In some embodiments, when pro-VWF is immobilized on an anion exchange resin and incubated with a solution that exhibits pro-VWF convertase activity, the conductivity measured at 25°C is less than 20 mS / cm. In some embodiments, when pro-VWF is immobilized on an anion exchange resin and incubated with a solution that exhibits pro-VWF convertase activity, the conductivity measured at 25°C is less than 16 mS / cm. In some embodiments, when pro-VWF is immobilized on an anion exchange resin and incubated with a solution that exhibits pro-VWF convertase activity, the conductivity measured at 25°C is between 16 mS / cm and 25 mS / cm. In some embodiments, when pro-rVWF is immobilized on an anion exchange resin and incubated with a solution exhibiting pro-VWF convertase activity, the conductivity measured at 25°C is 20 mS / cm to 25 mS / cm. Pro-rVWF and mat-rVWF can be efficiently immobilized on anion exchange resins at these conductivity levels. Therefore, buffers added during the method should be correspondingly adapted to maintain the conductivity level. In some embodiments, the conductivity is such that the furin and / or PACE enzymes are in active form and are present, fully or partially, in the mobile phase.

[0242] In some embodiments, mat-rVWF is eluted from the anion exchange resin at a conductivity of at least 40 mS / cm when measured at 25°C. In some embodiments, mat-rVWF is eluted from the anion exchange resin at a conductivity of at least 60 mS / cm when measured at 25°C. In some embodiments, mat-rVWF is eluted from the anion exchange resin at a conductivity of at least 80 mS / cm when measured at 25°C. In some embodiments, mat-rVWF is eluted from the anion exchange resin at a conductivity of 40 mS / cm to 80 mS / cm when measured at 25°C. In some embodiments, mat-rVWF is eluted from the anion exchange resin at a conductivity of 60 mS / cm to 80 mS / cm when measured at 25°C. In some embodiments, the desired rVWF species begins to elute using an anion exchange resin (e.g., using TMAE) at a conductivity of about 12 to 16 mS / cm at 25°C. In some embodiments, the bulk of the desired rVWF species is eluted using an anion exchange resin at about 55-60 mS / cm at 25°C. In some embodiments, the desired rVWF species begins to elute at a conductivity of about 18-24 mS / cm at 25°C using a cation exchange resin. In some embodiments, the bulk of the desired rVWF species is eluted using a cation exchange resin at about 36-42 mS / cm at 25°C. In some embodiments, the desired rVWF is mature rVWF (e.g., mat-rVWF). In some embodiments, the bulk comprises at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the total amount of the desired species eluted.

[0243] In some embodiments, an additional washing step is used before eluting mat-rVWF from the anion exchange resin. In some embodiments, an additional washing step is used before eluting mat-rVWF from the cation exchange resin.

[0244] Many proteases require cofactors, such as divalent metal ions, for their proteolytic activity. Furin and members of the furin protein family require calcium ions for activity. Therefore, when furin is used to mature pro-rVWF in vitro, a calcium salt is used. In some embodiments, the calcium salt is a soluble calcium salt. In some embodiments, the calcium salt is calcium chloride (CaCl). In some embodiments, the calcium salt is calcium acetate. In some embodiments, for example, Be 2+ , Ba 2+ , Mg 2+ , Mn 2+ , Sr 2+ , Zn 2+ , Co 2+ , Ni 2+ , Cd 2+ , and / or Cu 2+ Other divalent metal ions are used, including, but not limited to, Ca. In some embodiments, combinations of two or more divalent cations are used. In some embodiments, Ca 2+ and Mg 2+are used in combination. In some embodiments, the calcium salt is a soluble magnesium salt. In some embodiments, the magnesium salt is magnesium chloride (MgCl2). In some embodiments, a furin protein family formulation for use in maturation comprises a soluble calcium salt at a concentration of 0.01 to 10 mM. In some embodiments, a furin protein family formulation for use in maturation comprises a soluble magnesium salt at a concentration of 0.01 to 10 mM. In some embodiments, a furin protein family formulation for use in maturation comprises CaCl2 at a concentration of 0.01 to 10 mM. In some embodiments, a furin protein family formulation for use in maturation comprises MgCl2 at a concentration of 0.01 to 10 mM. In some embodiments, a furin protein family formulation for use in maturation comprises CaCl2 at a concentration of 0.1 to 5 mM. In some embodiments, a furin protein family formulation for use in maturation comprises MgCl2 at a concentration of 0.1 to 5 mM. In some embodiments, a furin protein family formulation for use in maturation comprises CaCl2 at a concentration of 0.2 to 2 mM. In some embodiments, the furin protein family preparation for use in maturation comprises a concentration of 0.2-2 mM MgCl. In some embodiments, the furin protein family preparation for use in maturation comprises furin. In some embodiments, the furin concentration is about 1, about 2, about 3, or about 4 units of recombinant active furin per IU of VWF:Ag (10 μg of pro-rVWF). In some embodiments, the furin concentration is about 2-3 units of recombinant active furin per IU of VWF:Ag (10 μg of pro-rVWF). In some embodiments, the furin concentration is about 1-2 units of recombinant active furin per IU of VWF:Ag (10 μg of pro-rVWF). In some embodiments, the furin concentration is about 2 units of recombinant active furin per IU of VWF:Ag (10 μg of pro-rVWF).

[0245] The incubation time of furin with immobilized pro-rVWF can vary depending on the system used. Factors such as temperature and buffer also affect the efficiency of the maturation process. Generally, the maturation process is complete within 48 hours. In some embodiments, the maturation process can occur in less than 1 minute. In some embodiments, the maturation process can occur in less than 40 hours, 36 hours, 30 hours, 24 hours, 20 hours, 16 hours, 10 hours, 5 hours, 2 hours, or 1 hour or less. In some embodiments, the incubation for pro-rVWF maturation is carried out for less than 1 minute to 48 hours. In some embodiments, the incubation for pro-rVWF maturation is carried out for 10 minutes to 42 hours. In some embodiments, the incubation for pro-rVWF maturation is carried out for 20 minutes to 36 hours. In some embodiments, the incubation for pro-rVWF maturation is carried out for 30 minutes to 24 hours. In some embodiments, the high specificity of furin prevents "hyperactivation" (further proteolysis) of VWF even after prolonged incubation.

[0246] In some embodiments, the maturation process also depends on the temperature selected during incubation, as the optimal enzymatic activity of furin varies with temperature.

[0247] In some embodiments, the incubation for pro-rVWF maturation is carried out at a temperature between 2° C. and 40° C. In some embodiments, the incubation for pro-rVWF maturation is carried out at a temperature between 4° C. and 37° C. In some embodiments, the incubation for pro-rVWF maturation is carried out at a lower temperature, for example, 2° C. In some embodiments, the maximum temperature used is lower than 50° C. to avoid and / or prevent proteolysis. In some embodiments, the maximum temperature used is lower than 45° C. to avoid and / or prevent proteolysis.

[0248] In some embodiments, pro-VWF (or pro-rVWF) is converted to mat-VWF (or mat-rVWF) by treatment with furin or a furin family member, as described above. In some embodiments, furin treatment converts at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% of pro-rVWF to mat-rVWF and rVWF-PP. In some embodiments, after size separation in the presence of the addition of at least one chelating agent and / or increasing the pH to at least pH 7, less than 5% rVWF-PP, less than 4% rVWF-PP, less than 3% rVWF-PP, less than 2% rVWF-PP, less than 1% rVWF-PP, less than 0.5% rVWF-PP, less than 0.4% rVWF-PP, less than 0.1% rVWF, or less than 0.05% rVWF-PP is present in the eluate.

[0249] Table 2. Exemplary pro-VWF removal (based on furin treatment) TIFF0007818898000002.tif46156

[0250] I. VWF multimers Assessment of the number and proportion of rVWF multimers can be performed using methods known in the art, including, but not limited to, methods for separating VWF multimers by size using electrophoresis and size-exclusion chromatography, as discussed by Cumming et al. (J. Clin. Pathol., 1993 May;46(5):470-473), which is incorporated herein by reference in its entirety for all purposes, particularly for all teachings related to the assessment of VWF multimers. Such techniques may further include immunoblotting techniques (such as Western blots) in which gels are immunoblotted with radiolabeled antibodies against VWF, followed by chemiluminescent detection (see, e.g., Wen et al., J. Clin. Lab. Anal., 1993, 7:317-323, which is incorporated herein by reference in its entirety for all purposes, particularly for all teachings related to the assessment of VWF multimers). Additional assays for VWF include VWF:antigen (VWF:Ag), VWF:ristocetin cofactor (VWF:RCof), and VWF:collagen binding activity assays (VWF:CBA), which are commonly used to diagnose and classify von Willebrand disease (see, e.g., Favaloro et al., Pathology, 1997, 29(4):341-456; Sadler, J.E., Annu Rev Biochem, 1998, 67:395-424; and Turecek et al., Semin Thromb Hemost, 2010, 36:510-521, which are incorporated by reference in their entirety for all purposes, particularly for all teachings related to assays for VWF). In some embodiments, the mat-rVWF obtained using the present methods comprises any multimer pattern present in the rVWF loading sample. In some embodiments, the mat-rVWF obtained using this method comprises physiologically occurring multimer patterns as well as extra-large VWF-multimer patterns.

[0251] J. VWF Assay In primary hemostasis, VWF functions as a bridge between platelets and certain components of the extracellular matrix, such as collagen. The biological activity of VWF in this process can be measured by various in vitro assays (Turecek et al., Semin Thromb Hemost, 2010, 36:510-521).

[0252] The VWF:ristocetin cofactor (VWF:RCof) assay is based on the aggregation of fresh or formalin-fixed platelets induced by the antibiotic ristocetin in the presence of VWF. The degree of platelet aggregation depends on the VWF concentration and can be measured using turbidimetric methods, such as an aggregometer (Weiss et al., J. Clin. Invest., 1973, 52:2708-2716; Macfarlane et al., Thromb. Diath. Haemorrh., 1975, 34:306-308). As provided herein, the specific ristocetin cofactor activity of the VWF (VWF:RCo) of the present invention, when measured using an in vitro assay, is generally described in terms of mU / μg of VWF.

[0253] In some embodiments, the mat-rVWF purified by the methods of the invention has a specific activity of at least about 20, 22.5, 25, 27, 5, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, 57.5, 60, 62.5, 65, 67.5, 70, 72.5, 75, 77.5, 80, 82.5, 85, 87.5, 90, 92.5, 95, 97.5, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, or more mU / μg. In some embodiments, the mat-rVWF used in the methods described herein has a specific activity of between 20 mU / μg and 150 mU / μg. In some embodiments, mat-rVWF has a specific activity of 30 mU / μg to 120 mU / μg. In some embodiments, mat-rVWF has a specific activity of 40 mU / μg to 90 mU / μg. In some embodiments, mat-rVWF has a specific activity selected from variations 1 to 133 found in Table 3 below.

[0254] Table 3: Exemplary embodiments of the specific activity of rVWF found in the compositions and used in the methods provided herein. TIFF0007818898000003.tif172144Var.=Variation

[0255] The mat-rVWF of the present invention is highly multimeric, containing about 10 to about 40 subunits. In further embodiments, the multimeric rVWF produced using the methods of the present invention contains about 10 to 30, 12 to 28, 14 to 26, 16 to 24, 18 to 22, or 20 to 21 subunits. In some embodiments, rVWF exists as multimers that vary in size from dimers to multimers of more than 40 subunits (>10 million daltons). The largest multimers provide multiple binding sites that can interact with both platelet receptors and subendothelial matrix sites of injury, and are the most hemostatically active form of VWF. In some embodiments, the mat-rVWF of the present invention includes ultra-large multimers (ULM). In general, high molecular weight and ultra-large multimers are believed to be most effective in terms of hemostasis (see, e.g., Turecek, P., Hamostaseologie, (Vol. 37): Supplement 1, pages S15-S25 (2017)). In some embodiments, mat-rVWF is between 500 kDa and 20,000 kDa. In some embodiments, any desired multimer pattern can be obtained using the described methods. In some embodiments, when using anion exchange and / or cation exchange methods, the pH, conductivity, and / or counterion concentration of the buffer in one or more wash steps or gradient buffers can be manipulated to obtain a desired multimer pattern. In some embodiments, size exclusion chromatography methods can be used to obtain a desired multimer pattern using recovery criteria. In some embodiments, the described multimer patterns include ultra-large multimers. In some embodiments, ultra-large multimers are at least 10,000 kDa, at least 11,000 kDa, at least 12,000 kDa, at least 13,000 kDa, at least 14,000 kDa, at least 15,000 kDa, at least 16,000 kDa, at least 17,000 kDa, at least 18,000 kDa, at least 19,000 kDa, or at least 20,000 kDa. In some embodiments, ultra-large multimers are between about 10,000 kDa and 20,000 kDa.In some embodiments, the ultra-large multimers are between about 11,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 12,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 13,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 14,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 15,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 16,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 17,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 18,000 kDa and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 19,000 kDa and 20,000 kDa. In some embodiments, the mat-rVWF obtained using the present methods includes any multimer pattern present in a loading sample of rVWF. In some embodiments, the mat-rVWF obtained using the present methods includes physiologically occurring multimer patterns and ultra-large VWF multimer patterns.

[0256] In some embodiments, the mat-rVWF composition prepared by the purification methods described herein has an rVWF oligomer distribution characterized in that 95% of the rVWF oligomers have between 6 and 20 subunits. In some embodiments, the mat-rVWF composition has an rVWF oligomer distribution characterized in that 95% of the rVWF oligomers have a range of subunits selected from the four observed variations of 458 to 641.

[0257] Table 4: Exemplary embodiments of rVWF oligomer distributions found in compositions and used in the methods provided herein. TIFF0007818898000004.tif82146TIFF0007818898000005.tif144146Var.=Variation

[0258] In some embodiments, the mat-rVWF compositions prepared by the methods provided herein can be characterized according to the percentage of mat-rVWF molecules present in specific higher-order mat-rVWF multimers or larger multimers. For example, in one embodiment, at least 20% of the mat-rVWF molecules in the mat-rVWF composition used in the methods described herein are present in oligomeric complexes of at least 10 subunits. In another embodiment, at least 20% of the mat-rVWF molecules in the mat-rVWF composition used in the methods described herein are present in oligomeric complexes of at least 12 subunits. In yet other embodiments, the mat-rVWF compositions used in the methods provided herein have a minimum percentage (e.g., at least X%) of mat-rVWF molecules present in specific higher-order mat-rVWF multimers or larger multimers (e.g., multimers of at least Y subunits) according to any one of variations 134-457 found in Tables 5-7.

[0259] (Table 5) Exemplary embodiments of the proportion of mat-rVWF molecules present in specific higher-order mat-rVWF multimers or larger multimers found in compositions and used in the methods provided herein. TIFF0007818898000006.tif98128Var.=Variation

[0260] (Table 6) Exemplary embodiments of the proportion of mat-rVWF molecules present in specific higher order mat-rVWF multimers or larger multimers found in compositions and used in the methods provided herein. TIFF0007818898000007.tif98128Var.=Variation

[0261] (Table 7) Exemplary embodiments of the proportion of mat-rVWF molecules present in specific higher order mat-rVWF multimers or larger multimers found in compositions and used in the methods provided herein. TIFF0007818898000008.tif96128Var.=Variation

[0262] In accordance with the above, mat-rVWF comprises a significant proportion of high molecular weight (HMW) mat-rVWF multimers. In further embodiments, the HMW rVWF multimer composition comprises at least 10%-80% mat-rVWF decamers or higher multimers. In further embodiments, the composition comprises about 10-95%, 20-90%, 30-85%, 40-80%, 50-75%, or 60-70% decamers or higher multimers. In further embodiments, the HMW mat-rVWF multimer composition comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% decamers or higher multimers.

[0263] For example, assessment of the number and proportion of mat-rVWF multimers can be performed using methods known in the art, including, but not limited to, methods for separating mat-rVWF multimers by size using electrophoresis and size-exclusion chromatography, as discussed by Cumming et al. (J Clin Pathol. 1993 May;46(5):470-473, which is incorporated herein by reference in its entirety for all purposes, particularly for all teachings related to the assessment of mat-rVWF multimers). Such techniques can further include immunoblotting (such as Western blot) using immunolabeling of gels with radiolabeled antibodies against VWF, followed by chemiluminescent detection (see, e.g., Wen et al. (1993), J. Clin. Lab. Anal., 7:317-323, which is incorporated herein by reference in its entirety for all purposes, particularly for all teachings related to the assessment of mat-rVWF multimers). Additional assays for VWF include the VWF:antigen (VWF:Ag), VWF:ristocetin cofactor (VWF:RCof), and VWF:collagen binding activity assay (VWF:CBA), which are commonly used in the diagnosis and classification of von Willebrand disease (see, e.g., Favaloro et al., Pathology, 1997, 29(4):341-456, which is incorporated herein by reference in its entirety for all purposes, particularly for all teachings related to assays for VWF).

[0264] In some embodiments, the ratio of rFVIII procoagulant activity (IU rFVIII:C) to rVWF ritosetin cofactor activity (IU rVWF:RCo) is 3:1 to 1:5 for mat-rVWF prepared according to the methods of the invention. In further embodiments, the ratio is 2:1 to 1:4. In yet further embodiments, the ratio is 5:2 to 1:4. In further embodiments, the ratio is 3:2 to 1:3. In still further embodiments, the ratio is about 1:1, 1:2, 1:3, 1:4, 1:5, 2:1, 2:3, 2:4, 2:5, 3:1, 3:2, 3:4, or 3:5. In further embodiments, the ratio is 1:1 to 1:2. In still further embodiments, the ratio is 1.1:1, 1.2:1, 1.3:1, 1.4:1, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1, or 2:1. In certain embodiments, the ratio of rFVIII procoagulant activity (IU rFVIII:C) to rVWF ritosetin cofactor activity (IU rVWF:RCo) in the compositions useful for the methods described herein is selected from variations 1988 to 2140 found in Table 8.

[0265] Table 8: Exemplary embodiments of the ratio of rFVIII procoagulant activity (IU rFVIII:C) to rVWF ritosetin cofactor activity (IU rVWF:RCo) in the compositions and for use in the methods provided herein. TIFF0007818898000009.tif213149TIFF0007818898000010.tif92149Var.=Variation

[0266] In further embodiments, the higher order mat-rVWF multimers of the present invention are stable for about 1 to about 90 hours after administration. In even further embodiments, the higher order mat-rVWF multimers are stable for about 5 to 80, 10 to 70, 15 to 60, 20 to 50, 25 to 40, or 30 to 35 hours after administration. In still further embodiments, the higher order mat-rVWF multimers are stable for at least 3, 6, 12, 18, 24, 36, 48, or 72 hours after administration. In certain embodiments, the stability of the mat-rVWF multimers is assessed in vitro.

[0267] In one embodiment, the higher order mat-rVWF multimers used in the compositions and methods provided herein have a half-life of at least 12 hours after administration. In another embodiment, the higher order mat-rVWF multimers have a half-life of at least 24 hours after administration. In yet another embodiment, the higher order mat-rVWF multimers have a half-life selected from variations 642 to 1045 found in Table 9.

[0268] Table 9: Exemplary embodiments of the half-life of higher order mat-rVWF multimers found in compositions prepared by the methods provided herein. TIFF0007818898000011.tif25136TIFF0007818898000012.tif220136TIFF0007818898000013.tif220136TIFF0007818898000014.tif25136Var.=Variation

[0269] In some embodiments, the pro-VWF and / or purified mat-rVWF purified according to the present invention is not modified by any complexation, post-translational modification, or covalent modification. In certain embodiments, the pro-VWF and / or purified mat-rVWF of the present invention is not modified with a water-soluble polymer, including but not limited to polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, polysialic acid, hydroxyethyl starch, polycarbohydrate moiety, etc.

[0270] In some embodiments, the purified pro-VWF and / or purified mat-rVWF according to the present invention are modified through conjugation, post-translational modification, or covalent modification, including modifications of the N- or C-terminal residues and selected side chains, such as free sulfhydryl groups, primary amines, and hydroxyl groups. In one embodiment, a water-soluble polymer is linked to the protein (directly or via a linker) through a lysine group or other primary amine. In some embodiments, the pro-VWF and / or purified mat-rVWF of the present invention may be modified by conjugation with a water-soluble polymer, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, polysialic acid, hydroxyethyl starch, polycarbohydrate moiety, etc.

[0271] Water-soluble polymers that can be used to modify pro-VWF and / or purified mat-rVWF include linear and branched structures. The conjugated polymers can be directly attached to the coagulation proteins of the present invention or can be attached via a linking moiety. Non-limiting examples of protein conjugation with water-soluble polymers can be found in U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; and 4,179,337, as well as Abuchowski and Davis, "Enzymes as Drugs," Holcenberg and Roberts, Eds., pp. 367-383, John Wiley & Sons, New York (1981), and Hermanson G., Bioconjugate Techniques, 2nd Ed., Academic Press, Inc. 2008.

[0272] Protein conjugation may be carried out by a number of techniques well known in the art; see, for example, Hermanson G., Bioconjugate Techniques 2nd Ed., Academic Press, Inc. 2008. Examples include linkage via a peptide bond between a carboxyl group on one side of the coagulation protein or the water-soluble polymer moiety and an amine group on the other, or an ester bond between a carboxyl group on one side and a hydroxyl group on the other. Another linkage that can conjugate the coagulation proteins of the present invention to water-soluble polymer compounds is via a Schiff base linkage between a free amino group on the polymer moiety that reacts with an aldehyde group formed at the non-reducing end of the polymer by periodate oxidation (Jennings and Lugowski, J. Immunol. 1981;127:1011-8; Femandes and Gregonradis, Biochim Biophys Acta. 1997;1341;26-34). The resulting Schiff base can be stabilized by specific reduction with NaCNBH3 to form a secondary amine. Another approach is the generation of terminal free amino groups on the polymer by reductive amination with NH4Cl after conventional oxidation. Bifunctional reagents can be used to link two amino groups or two hydroxyl groups. For example, a polymer containing an amino group can be coupled to the amino groups of a coagulation protein using a reagent such as BS3 (bis(sulfosuccinimidyl)suberate / Pierce, Rockford, Ill.). Furthermore, heterobifunctional cross-linking reagents such as sulfo-EMCS (N-ε-maleimidocaproyloxy) sulfosuccinimide ester / Pierce) can be used to link amine and thiol groups.In other embodiments, aldehyde-reactive groups, such as the diethyl acetal of PEG alkoxide and bromoacetaldehyde; PEG with DMSO and acetic anhydride; and phenoxide of PEG chloride and 4-hydroxybenzaldehyde, succinimidyl active ester, activated dithiocarbonate PEG, 2,4,5-trichlorophenyl chlorocarbonate, and p-nitrophenyl chlorocarbonate activated PEG, may be used for conjugation of coagulation proteins.

[0273] Another method for measuring the biological activity of VWF is the collagen binding assay based on ELISA technology (Brown and Bosak, Thromb. Res., 1986, 43:303-311; Favaloro, Thromb. Haemost., 2000, 83, 127-135). Microtiter plates are coated with type I or type III collagen. VWF is then bound to the collagen surface and subsequently detected with an enzyme-labeled polyclonal antibody. The final step is a substrate reaction that can be monitored photometrically in an ELISA reader.

[0274] Von Willebrand factor immunoassays (VWF:Ag) are immunoassays that measure the concentration of VWF protein in plasma. These do not indicate anything about VWF function. Many methods exist for measuring VWF:Ag, including both enzyme-linked immunosorbent assays (ELISAs) or automated latex immunoassays (LIAs). Many laboratories now use fully automated latex immunoassays. Historically, laboratories used a variety of techniques, including Laurel electroimmunoassays (Laurel Rockets), but these are rarely used in most laboratories today.

[0275] K. VWF preparation / administration The present invention also provides for the preparation of formulations from VWF obtained by the purification methods provided herein. In some embodiments, the high-purity mat-rVWF composition is used to prepare a pharmaceutical composition. In some embodiments, the mat-rVWF can be formulated into a lyophilized preparation.

[0276] In some embodiments, formulations containing the VWF polypeptides of the present invention are lyophilized after purification and before administration to a subject. Lyophilization can be performed using techniques common in the art and should be optimized for the composition under development (Tang et al., Pharm Res. 21:191-200, (2004) and Chang et al., Pharm Res. 13:243-9 (1996)).

[0277] In one embodiment, the lyophilization cycle consists of three steps: freezing, primary drying, and secondary drying (AP Mackenzie, Phil Trans R Soc London, Ser B, Biol 278:167 (1977)). The freezing step cools the solution to initiate ice formation. This step also induces crystallization of the bulking agent. Sublimation of the ice in the primary drying step is achieved by using vacuum to reduce the chamber pressure below the vapor pressure of the ice and introducing heat to promote sublimation. Finally, adsorbed or bound water is removed in the secondary drying step at elevated shelf temperatures under reduced chamber pressure. This process produces a material known as a lyophilized cake. The cake can then be reconstituted with either sterile water or a suitable diluent for injection.

[0278] The lyophilization cycle not only determines the final physical state of an excipient, but also influences other parameters such as reconstitution time, appearance, stability, and final moisture content. The compositional structure of the frozen state progresses through several transitions (e.g., glass transition, wetting, and crystallization) that occur at specific temperatures, and this structure may be used to understand and optimize the lyophilization process. The glass transition temperature (Tg and / or Tg') can provide information about the physical state of the solute and can be measured by differential scanning calorimetry (DSC). Tg and Tg' are important parameters that must be considered when designing a lyophilization cycle. For example, Tg' is important for primary drying. Furthermore, in the dried state, the glass transition temperature provides information about the storage temperature of the final product.

[0279] i. General Pharmaceutical Formulations and Excipients Excipients are additives that confer or enhance the stability and delivery of pharmaceutical formulations (e.g., proteins). Regardless of the reason for their inclusion, excipients are essential components of the formulation and therefore must be safe and well tolerated by patients. For protein drugs, the choice of excipient is particularly important because it can affect both the efficacy and immunogenicity of the drug. Therefore, protein formulations must be developed with the appropriate selection of excipients that provide adequate stability, safety, and marketability.

[0280] In one embodiment, the lyophilized formulation comprises at least one or more buffers, bulking agents, and stabilizers. In this embodiment, surfactants are evaluated and selected for their usefulness when aggregation is an issue during the lyophilization process or reconstitution. Appropriate buffers are included to maintain the formulation within a stable pH zone during lyophilization. A comparison of excipient components for liquid and lyophilized protein formulations is provided in Table 10.

[0281] Table 10. Excipient components of lyophilized protein formulations TIFF0007818898000015.tif228117

[0282] A major challenge in developing protein formulations is stabilizing the product against the stresses of manufacturing, shipping, and storage. The role of formulation excipients is to provide stabilization against these stresses. Excipients are also used to reduce the viscosity of highly concentrated protein formulations to enable their delivery and enhance patient convenience. Generally, excipients can be classified based on the mechanism by which they stabilize proteins against various chemical and physical stresses. Some excipients are used to mitigate the effects of specific stresses or to modulate specific susceptibility of a particular protein. Other excipients have a more general effect on the physical and covalent stability of proteins. The excipients described herein are organized by their chemical type or their functional role in the formulation. A brief description of the mode of stabilization is provided when discussing each excipient type.

[0283] Given the teachings and guidance provided herein, one of skill in the art will know the amounts or ranges of excipients that can be included in any particular formulation to achieve a biopharmaceutical formulation of the invention that will promote the maintenance of stability of the biopharmaceutical (e.g., protein). For example, the amount and type of salt to include in a biopharmaceutical formulation of the invention is selected based on the desired tonicity (e.g., isotonic, hypotonic, or hypertonic) of the final solution and the amounts and tonicities of other components included in the formulation.

[0284] For example, isotonicity can be achieved by including about 5% sorbitol, while about 9% sucrose excipient is required to achieve isotonicity. The selection of the amount or range of concentrations of one or more excipients that can be included in the biopharmaceutical formulations of the invention is exemplified above with reference to salts, polyols, and sugars. However, those skilled in the art will understand that the considerations described herein, and further exemplified with reference to specific excipients, are equally applicable to all types and combinations of excipients, including, for example, salts, amino acids, other isotonicity agents, surfactants, stabilizers, bulking agents, cryoprotectants, lyoprotectants, antioxidants, metal ions, chelating agents, and / or protectants.

[0285] Additionally, when reporting a particular excipient in molar concentration, one of skill in the art will recognize that the equivalent weight (%) w / v of the solution (e.g., (grams of substance in solution sample / mL solution) x 100%) is also assumed.

[0286] Of course, those skilled in the art will recognize that the concentrations of excipients described herein share interdependencies within a particular formulation. For example, the concentration of a bulking agent may be reduced, for example, in the case of a high protein concentration, or, for example, in the case of a high stabilizer concentration. Furthermore, those skilled in the art will recognize that to maintain the isotonicity of a particular formulation without a bulking agent, the concentration of the stabilizer will be adjusted accordingly (e.g., a "tonicity-increasing" amount of stabilizer will be used). Common excipients are known in the art and can be found in Powell et al., Compendium of Excipients for Parenteral Formulations (1998), PDA J. Pharm. Sci. Technology, 52:238-311.

[0287] ii. Pharmaceutical buffers and buffering agents The stability of pharmacologically active protein formulations is usually observed to be greatest within a narrow pH range. This pH range of optimal stability needs to be identified early during preformulation studies. Several approaches, such as accelerated stability studies and calorimetric screening studies, are useful in this effort (Remmele RL Jr., et al., Biochemistry, 38(16):5241-7(1999)). Once the formulation is established, the protein must be manufactured and maintained throughout its shelf life. Therefore, buffers are most often used to control the pH in the formulation.

[0288] The buffering capacity of a buffer species is greatest at a pH equal to its pKa and decreases as the pH moves away from this value. 90% of the buffering capacity is within one pH unit of its pKa. Buffering capacity increases proportionally with increasing buffer concentration.

[0289] Several factors must be considered when selecting a buffer. First, the buffer species and its concentration must be defined based on its pKa and the desired formulation pH. Similarly, it is important to ensure that the buffer is compatible with proteins and other formulation excipients and does not catalyze degradation reactions. A third important aspect to consider is the stinging and irritation sensation that the buffer may induce upon administration. For example, citric acid is known to cause stinging upon injection (Laursen T, et al., Basic Clin Pharmacol Toxicol., 98(2):218-21(2006)). The potential for stinging and irritation is greater for drugs administered via the subcutaneous (SC) or intramuscular (IM) routes, where the drug solution remains in place for a relatively longer period than when administered via an IV route, where the formulation is rapidly diluted in the blood upon administration. For formulations administered via direct IV infusion, the total amount of buffer (and other formulation components) must be monitored. Particular caution should be exercised regarding potassium ions administered in the form of potassium phosphate buffer, which can induce cardiovascular effects in patients (Hollander-Rodriguez JC, et al., Am. Fam. Physician., 73(2):283-90 (2006)).

[0290] Buffers for lyophilized formulations require additional consideration. Some buffers, such as sodium phosphate, crystallize from the protein amorphous phase during freezing, resulting in a pH shift. Other common buffers, such as acetic acid and imidazole, can sublimate or evaporate during the lyophilization process, thereby shifting the pH of the formulation during lyophilization or after reconstitution.

[0291] The buffer system present in the composition is physiologically compatible and is selected to maintain the desired pH of the pharmaceutical formulation. In one embodiment, the pH of the solution is between pH 2.0 and pH 12.0. For example, the pH of the solution may be 2.0, 2.3, 2.5, 2.7, 3.0, 3.3, 3.5, 3.7, 4.0, 4.3, 4.5, 4.7, 5.0, 5.3, 5.5, 5.7, 6.0, 6.3, 6.5, 6.7, 7.0, 7.3, 7.5, 7.7, 8.0, 8.3, 8.5, 8.7, 9.0, 9.3, 9.5, 9.7, 10.0, 10.3, 10.5, 10.7, 11.0, 11.3, 11.5, 11.7, or 12.0.

[0292] The pH buffering compound may be present in any amount suitable for maintaining the pH of the formulation at a predetermined level. In one embodiment, the pH buffer concentration is 0.1 mM to 500 mM (1 M). For example, it is contemplated that the pH buffering agent may be at least 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 mM.

[0293] Exemplary pH buffering agents used to buffer the formulations described herein include, but are not limited to, organic acids, glycine, histidine, glutamate, succinate, phosphate, acetate, citrate, Tris, HEPES, and amino acids or mixtures of amino acids, including, but not limited to, aspartate, histidine, and glycine. In one embodiment of the present invention, the buffering agent is citrate.

[0294] In some embodiments, the formulation comprises 50 mM glycine, 10 mM taurine, 5% (w / w) sucrose, 5% (w / w) D-mannitol, 0.1% polysorbate 80, 2 mM CaCl, 150 mM NaCl, and has a pH of 7.4. In some embodiments, the formulation comprises highly purified mat-rVWF, 50 mM glycine, 10 mM taurine, 5% (w / w) sucrose, 5% (w / w) D-mannitol, 0.1% polysorbate 80, 2 mM CaCl, 150 mM NaCl, and has a pH of 7.4. In some embodiments, the formulation comprises vWF and / or r-vWF / rFVIII and 50 mM glycine, 10 mM taurine, 5% (w / w) sucrose, 5% (w / w) D-mannitol, 0.1% polysorbate 80, 2 mM CaCl2, 150 mM NaCl, and has a pH of 7.4.

[0295] iii. Pharmaceutical stabilizers and bulking agents In one embodiment of the pharmaceutical formulation, a stabilizer (or a combination of stabilizers) is added to prevent or reduce storage-induced aggregation and chemical degradation. A hazy or cloudy solution upon reconstitution indicates that the protein has precipitated or at least aggregated. The term "stabilizer" refers to an excipient that can prevent aggregation or physical deterioration, including chemical degradation (e.g., autolysis, deamidation, oxidation, etc.) in aqueous conditions. Contemplated stabilizers include, but are not limited to, polyhydroxy compounds, including sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, glycine, arginine HCl, dextran, starch, hydroxyethyl starch, cyclodextrin, N-methylpyrrolidine, cellulose, and polysaccharides such as sodium hyaluronate (Carpenter et al., Develop. Biol. Standard 74:225, (1991)). The formulations include stabilizers at a concentration of about 0.1, 0.5, 0.7, 0.8 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, 900, or 1000 mM. In one embodiment of the invention, mannitol and trehalose are used as stabilizers.

[0296] Optionally, the formulation also contains an appropriate amount of a bulking agent and an osmolality adjusting agent. Examples of bulking agents include, but are not limited to, mannitol, glycine, sucrose, polymers such as dextran, polyvinylpyrrolidone, carboxymethylcellulose, lactose, sorbitol, trehalose, or xylitol. In one embodiment, the bulking agent is mannitol. The bulking agent is present at a concentration of about 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, 900, or 1000 mM.

[0297] iv. Pharmaceutical surfactants Proteins have a high tendency to interact with surfaces, making them susceptible to adsorption and denaturation at air-liquid, vial-liquid, and liquid-liquid (silicone oil) interfaces. This degradation pathway has been observed to be inversely proportional to protein concentration and results from the loss of protein from solution due to the formation of soluble and insoluble protein aggregates or adsorption to surfaces. In addition to container surface adsorption, surface-induced degradation is exacerbated by physical agitation that may be experienced during product shipping and handling.

[0298] Surfactants are commonly used in protein formulations to prevent surface-induced degradation. Surfactants are amphiphilic molecules that have the ability to compete with proteins for interfacial positions. The hydrophobic portion of the surfactant molecule occupies interfacial positions (e.g., air / liquid), while the hydrophilic portion of the molecule remains oriented toward the bulk solvent. At sufficient concentrations (usually near the surfactant's critical micelle concentration), the surface layer of surfactant molecules serves to prevent protein molecules from adsorbing at the interface, thereby minimizing surface-induced degradation. Surfactants contemplated herein include, but are not limited to, fatty acid esters of sorbitan polyethoxylate, such as polysorbate 20 and polysorbate 80. These two surfactants differ only in the length of the aliphatic chain, C-12 and C-18, respectively, which confers hydrophobic character to the molecule. Thus, polysorbate-80 has higher surface activity and a lower critical micelle concentration than polysorbate-20.

[0299] Detergents can also affect the thermodynamic conformational stability of proteins. Again, the effect of a given surfactant excipient is protein-specific. For example, polysorbates have been shown to decrease the stability of some proteins and increase the stability of others. Protein destabilization by detergents can be rationalized in terms of the hydrophobic tails of detergent molecules, which can engage in specific binding with partially or fully unfolded protein states. These types of interactions can cause a shift in conformational equilibrium toward more extended protein states (e.g., increasing the exposure of hydrophobic portions of protein molecules that complement bound polysorbates). Alternatively, if the native state of a protein exhibits some hydrophobic surface, detergent binding to the native state can stabilize that conformation.

[0300] Another aspect of polysorbates is that they are inherently susceptible to oxidative degradation. Often, as raw materials, they contain sufficient amounts of peroxides to cause oxidation of protein residue side chains, especially methionine. The possibility of oxidative damage due to the addition of stabilizers emphasizes the need to use the lowest effective concentration of excipients in formulations. For surfactants, the effective concentration for a given protein depends on the stabilization mechanism.

[0301] In addition, surfactants are added in appropriate amounts to prevent surface-related aggregation phenomena during freezing and drying (Chang, B, J. Pharm. Sci. 85:1325, (1996)). Accordingly, exemplary surfactants include anionic, cationic, nonionic, zwitterionic, and amphoteric surfactants, including surfactants derived from natural amino acids. Anionic surfactants include, but are not limited to, sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate, chenodeoxycholic acid, N-lauroyl sarcosine sodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate, and glycodeoxycholic acid sodium salt. Cationic surfactants include, but are not limited to, benzalkonium chloride or benzethonium chloride, cetylpyridinium chloride monohydrate, and hexadecyltrimethylammonium bromide. Zwitterionic surfactants include, but are not limited to, CHAPS, CHAPSO, SB3-10, and SB3-12. Nonionic surfactants include, but are not limited to, digitonin, Triton X-100, Triton X-114, TWEEN-20, and TWEEN-80. Surfactants also include, but are not limited to, lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50, and 60, glycerol monostearate, polysorbate 40, 60, 65, and 80, soybean lecithin, and other phospholipids such as dioleylphosphatidylcholine (DOPC), dimyristoylphosphatidylglycerol (DMPG), dimyristoylphosphatidylcholine (DMPC), and (dioleylphosphatidylglycerol)DOPG; sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Therefore, compositions containing these surfactants individually or as a mixture in different ratios are further provided. In one embodiment of the present invention, the surfactant is TWEEN-80. In this formulation, the surfactant is present at a concentration of about 0.01 to about 0.5 g / L.In the provided formulations, the surfactant concentration is 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 or 1.0 g / L.

[0302] v. Pharmaceutical salts Salts are often added to increase the ionic strength of a formulation, which can be important for protein solubility, physical stability, and isotonicity. Salts can affect the physical stability of proteins in various ways. Ions can stabilize the native state of a protein by binding to charged residues on the protein surface. Alternatively, salts can stabilize the denatured state by binding to peptide groups along the protein backbone (-CONH-). Salts can also stabilize the native conformation of a protein by shielding repulsive electrostatic interactions between residues within the protein molecule. Salts in protein formulations can also shield attractive electrostatic interactions between protein molecules, which can lead to protein aggregation and insolubility. In the provided formulations, the salt concentration is between 0.1, 1, 10, 20, 30, 40, 50, 80, 100, 120, 150, 200, 300, and 500 mM.

[0303] vi. Other common excipient ingredients: pharmaceutical amino acids Amino acids are widely used in protein formulations as buffers, bulking agents, stabilizers, and antioxidants. Thus, in one embodiment, histidine and glutamic acid are used to buffer protein formulations in the pH ranges of 5.5-6.5 and 4.0-5.5, respectively. The imidazole group of histidine has a pKa of 6.0, and the carboxyl group of the glutamic acid side chain has a pKa of 4.3, making these amino acids suitable for buffering in their respective pH ranges. Glutamic acid is particularly useful in such cases. Histidine is commonly found in commercial protein formulations, and this amino acid is an alternative to citrate, a buffer known for its stinging properties upon injection. Interestingly, histidine has also been reported to have a stabilizing effect on aggregation when used at high concentrations in both liquid and lyophilized presentations (Chen B, et al., Pharm Res., 20(12):1952-60 (2003)). Another report also observed that histidine reduced the viscosity of high-protein-concentration formulations. However, in the same study, the authors observed increased aggregation and discoloration of histidine-containing formulations during freeze-thaw studies of antibodies in stainless steel containers. Another caveat with histidine is that it undergoes photooxidation in the presence of metal ions (Tomita M, et al., Biochemistry, 8(12):5149-60(1969)). The use of methionine as an antioxidant in formulations appears promising; it has been observed to be effective against many oxidative stresses (Lam XM, et al., J Pharm ScL, 86(11):1250-5(1997)).

[0304] In various embodiments, formulations are provided that include one or more of the amino acids glycine, proline, serine, arginine, and alanine, which have been shown to stabilize proteins by a preferential exclusion mechanism. Glycine is also a commonly used bulking agent in lyophilized formulations. Arginine has been shown to be an effective agent in inhibiting aggregation and is used in both liquid and lyophilized formulations.

[0305] In the provided formulations, the amino acid concentration is between 0.1, 1, 10, 20, 30, 40, 50, 80, 100, 120, 150, 200, 300 and 500 mM. In one embodiment of the invention, the amino acid is glycine.

[0306] vii. Other Common Excipient Ingredients: Pharmaceutical Antioxidants Oxidation of protein residues occurs from many different sources. In addition to the addition of specific antioxidants, preventing oxidative protein damage involves careful control of many factors throughout the product manufacturing process and storage, including atmospheric oxygen, temperature, light exposure, and chemical contamination. Therefore, the present invention contemplates the use of pharmaceutical antioxidants, including, but not limited to, reducing agents, oxygen / free radical scavengers, or chelating agents. Antioxidants in therapeutic protein formulations, in one embodiment, are water-soluble and maintain activity throughout the product's shelf life. Reducing agents and oxygen / free radical scavengers act by scavenging reactive oxygen species in solution. Chelating agents, such as EDTA, work by binding trace metal contaminants that promote free radical formation. For example, EDTA was utilized in a liquid formulation of acidic fibroblast growth factor to inhibit metal ion-catalyzed oxidation of cysteine ​​residues.

[0307] In addition to the effectiveness of various excipients in preventing protein oxidation, there is concern that antioxidants themselves may induce other covalent or physical changes in proteins. For example, reducing agents can cause intramolecular disruption of disulfide bonds, leading to disulfide shuffling. In the presence of transition metal ions, ascorbic acid and EDTA have been shown to promote methionine oxidation in many proteins and peptides (Akers MJ, and Defelippis MR. Peptides and Proteins as Parenteral Solutions. In: Pharmaceutical Formulation Development of Peptides and Proteins. Sven Frokjaer, Lars Hovgaard, editors. Pharmaceutical Science. Taylor and Francis, UK (1999)); Fransson JR, / . Pharm. Sci. 86(9):4046-1050 (1997); Yin J, et al., Pharm Res., 21(12):2377-83 (2004)). Sodium thiosulfate has been reported to reduce the level of light- and temperature-induced methionine oxidation in rhuMab HER2; however, this study also reported the formation of thiosulfate-protein adducts (Lam XM, Yang JY, et al., J Pharm Sci. 86(11):1250-5(1997)). The selection of an appropriate antioxidant depends on the specific stress and sensitivity of the protein. Antioxidants contemplated in certain embodiments include, but are not limited to, reducing agents and oxygen / free radical scavengers, EDTA, and sodium thiosulfate.

[0308] viii. Other common excipient ingredients: pharmaceutical metal ions Generally, transition metal ions are undesirable in protein formulations because they can catalyze physical and chemical decomposition reactions in proteins. However, certain metal ions are included in protein formulations when they are cofactors and when they form coordination complexes in protein suspension formulations (e.g., zinc suspensions of insulin). Recently, the use of magnesium ions (10-120 mM) has been proposed to inhibit the isomerization of aspartic acid to isoaspartic acid (WO2004039337).

[0309] Two examples where metal ions confer stability or enhance activity to proteins are human deoxyribonuclease (rhDNase, Pulmozyme®) and Factor VIII. In the case of rhDNase, Ca +2 ions (up to 100 mM) increased the stability of the enzyme through specific binding sites (Chen B, et al., / Pharm Sci., 88(4):477-82(1999)). Indeed, removal of calcium ions from the solution using EGTA caused increased deamidation and aggregation. However, this effect was not observed in the presence of Ca +2 ion; other divalent cations Mg +2 , Mn +2 and Zn +2 Ca was observed to destabilize rhDNase. A similar effect was observed with factor VIII. +2 and Sr +2 ions stabilize proteins, while Mg+ 2 , Mn +2 and Zn +2 , Cu +2 and Fe +2 Other ions such as Al destabilized the enzyme (Fatouros, A., et al., Int. J. Pharm., 155, 121-131 (1997)). In another study using factor VIII, Al +3 A significant increase in the rate of aggregation was observed in the presence of ions (Derrick TS, et al., / . Pharm. Sci., 93(10):2549-57(2004)). The authors also found that other excipients, such as buffer salts, could increase the aggregation rate.+3 It has been pointed out that pharmaceuticals are often contaminated with ions, highlighting the need to use excipients of appropriate quality in formulated products.

[0310] ix. Other Common Excipient Ingredients: Pharmaceutical Preservatives Preservatives are necessary when developing multi-use parenteral formulations involving multiple extractions from the same container. Their primary function is to inhibit microbial growth and ensure product sterility throughout the drug's shelf life or usage period. Commonly used preservatives include, but are not limited to, benzyl alcohol, phenol, and m-cresol. Although preservatives have been used for a long time, developing protein formulations containing preservatives can be challenging. Preservatives almost always have a destabilizing effect on proteins (aggregation), which is a major factor limiting their use in multi-dose protein formulations (Roy S, et al., J Pharm ScL, 94(2):382-96(2005)).

[0311] To date, most protein pharmaceuticals have been formulated for single use only. However, where multi-dose formulations are possible, they offer the added benefit of enabling patient convenience and increasing marketability. A good example is that of human growth hormone (hGH), where the development of preserved formulations has led to the commercialization of more convenient and versatile injection pen presentations. At least four such pen devices containing preserved formulations of hGH are currently available on the market. Norditropin® (liquid, Novo Nordisk), Nutropin AQ® (liquid, Genentech), and Genotropin (lyophilized dual-chamber cartridge, Pharmacia & Upjohn) contain phenol, while Somatrope® (Eli Lilly) is formulated with m-cresol.

[0312] During formulation development of a preserved dosage form, several aspects must be considered. The effective preservative concentration of a pharmaceutical product must be optimized. This requires testing a given preservative in the dosage form over a range of concentrations that provide antimicrobial efficacy without compromising protein stability. For example, in the development of a liquid formulation of interleukin-1 receptor (type I), three preservatives were successfully screened using differential scanning calorimetry (DSC). The preservatives were ranked based on their impact on stability at concentrations commonly used in commercial products (Remmele RL Jr., et al., PharmRes., 15(2):200-8(1998)).

[0313] Developing liquid formulations containing preservatives is more challenging than lyophilized formulations. Lyophilized products can be lyophilized without preservatives and reconstituted with a preservative-containing diluent at the time of use. This significantly reduces the time the preservative is in contact with the protein, minimizing associated stability risks. In liquid formulations, the preservative's effectiveness and stability must be maintained throughout the product's shelf life (18-24 months). An important point to note is that preservative effectiveness must be demonstrated in the final formulation containing the active drug and all excipient components.

[0314] Some preservatives may cause injection site reactions, which is another factor that needs to be considered when selecting a preservative. In a clinical trial focused on evaluating preservatives and buffers for Norditropin, pain perception was observed to be lower in formulations containing phenol and benzyl alcohol compared to formulations containing m-cresol (Kappelgaard AM, Horm Res. 62 Suppl 3:98-103 (2004)). Interestingly, among commonly used preservatives, benzyl alcohol has anesthetic properties (Minogue SC, and Sun DA., AnesthAnalg., 100(3):683-6 (2005)). In various embodiments, the use of a preservative provides benefits that outweigh any side effects.

[0315] x. Methods for preparing pharmaceutical preparations The present invention further contemplates a method for preparing a pharmaceutical formulation.

[0316] The method further comprises one or more of the following steps: adding a stabilizer as described herein to the mixture before freeze-drying; and adding at least one agent selected from a bulking agent, an osmotic agent, and a surfactant, each as described herein, to the mixture before freeze-drying.

[0317] Standard reconstitution practice for lyophilized material is to add a certain amount of purified or sterile water for injection (WFI) (usually equivalent to the volume removed during lyophilization), although dilute solutions of antibacterial agents may also be used in the manufacture of pharmaceuticals for parenteral administration (Chen, Drug Development and Industrial Pharmacy, 18:1311-1354 (1992)). Accordingly, a method for preparing a reconstituted rVWF composition of the present invention is provided, which comprises adding a diluent to the lyophilized rVWF composition.

[0318] The lyophilized material may be reconstituted as an aqueous solution using a variety of aqueous carriers, for example, sterile water for injection, water containing a preservative for multiple dose use, or water containing an appropriate amount of surfactant (e.g., aqueous suspensions containing the active compound in admixture with excipients suitable for the manufacture of aqueous suspensions). In various embodiments, such excipients include suspending agents, such as, but not limited to, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum acacia; dispersing or wetting agents include natural phosphatides, such as, but not limited to, lecithin, or condensation products of alkylene oxides with fatty acids, such as, but not limited to, polyoxyethylene stearate, or condensation products of ethylene oxide with long-chain aliphatic alcohols, such as, but not limited to, heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitols, such as, but not limited to, polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with fatty acids and partial esters derived from hexitol anhydrides, such as, but not limited to, polyethylene sorbitan monooleate. In various embodiments, aqueous suspensions also include one or more preservatives, such as, but not limited to, ethyl, n-propyl, p-hydroxybenzoate.

[0319] xi. Exemplary mat-rVWF Formulations for Administration In some embodiments, the method provides an enhanced formulation (100 IU / ml to 10,000 IU / ml) such that the final product may have high potency (high mat-rVWF concentration and enhanced long-term stability) to reduce the amount of therapeutic agent. In some embodiments, the mat-rVWF concentration in the formulation for administration is about 100 IU / ml to 10,000 IU / ml. In some embodiments, the mat-rVWF concentration in the formulation for administration is about 500 IU / ml to 10,000 IU / ml. In some embodiments, the mat-rVWF concentration in the formulation for administration is about 1,000 IU / ml to 10,000 IU / ml. In some embodiments, the mat-rVWF concentration in the formulation for administration is about 2,000 IU / ml to 10,000 IU / ml. In some embodiments, the mat-rVWF concentration in the formulation for administration is about 3,000 IU / ml to 10,000 IU / ml. In some embodiments, the mat-rVWF concentration in the formulation for administration is about 4000 IU / ml to 10000 IU / ml. In some embodiments, the mat-rVWF concentration in the formulation for administration is about 5000 IU / ml to 10000 IU / ml. In some embodiments, the mat-rVWF concentration in the formulation for administration is about 6000 IU / ml to 10000 IU / ml. In some embodiments, the mat-rVWF concentration in the formulation for administration is about 7000 IU / ml to 10000 IU / ml. In some embodiments, the mat-rVWF concentration in the formulation for administration is about 8000 IU / ml to 10000 IU / ml. In some embodiments, the mat-rVWF concentration in the formulation for administration is about 9000 IU / ml to 10000 IU / ml. In some embodiments, mat-rVWF is formulated with recombinant coagulation factor VIII (rFVIII). In some embodiments, the rFVIII is full-length FVIII. In some embodiments, the rFVIII is full-length and chemically modified. In some embodiments, the rFVIII includes a FVIII fusion protein having a FIX activation peptide in place of the B domain. In some embodiments, the rFVIII is a FVIII hybrid with a truncated glycosylation-rich B domain.In some embodiments, the FVIII is a FVIII B-domain deleted variant. In some embodiments, the FVIII is a chemically modified variant of a FVIII B-domain deleted variant. In some embodiments, mat-rVWF is formulated with rFVIII prior to the lyophilization or end-of-fill steps, and the components are preserved by mixing in vitro or in an "on-column" procedure (e.g., adding FVIII during the purification process).

[0320] In some embodiments, the formulation for administration contains one or more zwitterionic compounds, including, for example, amino acids such as histidine, glycine, and arginine. In some embodiments, the formulation for administration contains a component with amphiphilic properties, having at least one hydrophobic group and one hydrophilic group, including, for example, polysorbate 80, octylpyranoside, dipeptides, and / or amphipathic peptides. In some embodiments, the formulation for administration contains a non-reducing sugar or sugar alcohol or disaccharide, including, for example, sorbitol, mannitol, sucrose, or trehalose. In some embodiments, the formulation for administration contains a non-toxic water-soluble salt that provides physiological osmolality, including, for example, sodium chloride. In some embodiments, the formulation for administration has a pH in the range of 6.0 to 8.0. In some embodiments, the formulation for administration has a pH of about 6.0, about 6.5, about 7, about 7.5, or about 8.0. In some embodiments, the formulation for administration contains one or more divalent cations that stabilize rVWF, including, for example, Ca2+, Mg2+, Zn2+, Mn2+, and / or combinations thereof. In some embodiments, the formulation for administration contains about 1 mM to about 50 mM glycine, about 1 mM to about 50 mM histidine, about 0 mM to about 300 mM sodium chloride (e.g., less than 300 mM sodium), about 0.01% to about 0.05% polysorbate 20 (or polysorbate 80), and about 0.5% to about 20% (w / w) sucrose, and has a pH of about 7.0 and a physiological osmolality at the time of administration.

[0321] In some embodiments, the formulation for administration can be lyophilized. In some embodiments, the formulation for administration is stable and can be stored in liquid form at about 2°C to about 8°C, and about 18°C ​​to about 25°C. In some embodiments, the formulation for administration is stable and can be stored in liquid form at about 2°C to about 8°C. In some embodiments, the formulation for administration is stable and can be stored in liquid form at about 18°C ​​to about 25°C.

[0322] xii. Administration For administration of a composition to a human or animal subject, in one embodiment, the composition comprises one or more pharmaceutically acceptable carriers. The terms "pharmaceutically" or "pharmacologically" acceptable refer to molecular entities and compositions that are stable, inhibit proteolytic degradation, including aggregation and cleavage products, and do not cause allergic or other adverse reactions when administered by routes well known in the art, as described below. "Pharmaceutically acceptable carriers" include any and all physiologically compatible, clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, for example, those disclosed above.

[0323] Pharmaceutical preparations are administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or by intracranial injection. As used herein, the term "parenterally" includes subcutaneous injection, intravenous, intramuscular, and intrasternal injection or infusion techniques. Administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, and intrapulmonary injection, and / or surgical implantation at a specific site are also contemplated. Generally, compositions are essentially free of pyrogens and other impurities that may be harmful to the recipient.

[0324] Single or multiple administrations of the composition are carried out with the dose level and pattern being selected by the treating physician. For the prevention or treatment of disease, the appropriate dose will depend on the type of disease being treated, as defined above, the severity and course of the disease, whether the drug is being administered prophylactically or therapeutically, previous treatments, the patient's medical history and response to the drugs, and the discretion of the treating physician.

[0325] xiii. Kit In a further aspect, the present invention includes kits containing one or more lyophilized compositions packaged in a manner that facilitates their use for administration to a subject. In one embodiment, such a kit includes a pharmaceutical formulation described herein (e.g., a composition comprising a therapeutic protein or peptide) packaged in a container, e.g., a sealed bottle or container, along with a label affixed to the container or included in the package that describes the use of the compound or composition in practicing a method. In one embodiment, the pharmaceutical formulation is packaged in a container such that the amount of headspace in the container (e.g., the amount of air between the liquid formulation and the top of the container) is very small. Preferably, the amount of headspace is negligible (e.g., virtually none). In one embodiment, the kit includes a first container containing a therapeutic protein or peptide composition and a second container containing a physiologically acceptable reconstitution solution for the composition. In one aspect, the pharmaceutical formulation is packaged in a unit dosage form. The kit may further include a device suitable for administering the pharmaceutical formulation according to a specific route of administration. Preferably, the kit includes a label describing the use of the pharmaceutical formulation.

[0326] xiv.Dose The dosage regimen for the methods of treating the conditions described herein will be determined by the attending physician, taking into account various factors that modify the drug's effect, such as the patient's age, condition, weight, sex, and diet, the severity of the infection, the time of administration, and other clinical factors. For example, a typical dose of the recombinant VWF of the present invention is approximately 50 U / kg, which is equivalent to 500 μg / kg.

[0327] In one embodiment, the formulations of the present invention are administered by an initial bolus followed by a continuous infusion to maintain therapeutic circulating levels of the formulation. In another example, the compounds of the present invention are administered as a single dose. Those skilled in the art will readily optimize effective dosages and administration schedules, as determined by good medical practice and the clinical condition of each individual patient. The frequency of administration depends on the pharmacokinetic parameters of the drug and the route of administration. The optimal pharmaceutical formulation is determined by those skilled in the art depending on the route of administration and the desired dose. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712, the disclosure of which is incorporated herein by reference. Such formulations affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the administered drug. Depending on the route of administration, appropriate doses are calculated according to body weight, body surface area, or organ size. Appropriate doses may be confirmed using established assays for measuring blood concentrations of the dose in conjunction with appropriate dose-response data. The final dosing regimen will be determined by the attending physician, taking into account various factors that modify the drug's action, such as the specific activity of the drug, the severity and responsiveness of the patient's injury, the patient's age, condition, weight, sex, and diet, the severity of the infection, the time of administration, and other clinical factors. As research continues, more information will become available regarding appropriate dose levels and duration of treatment for various diseases and conditions. [Example]

[0328] The following non-limiting examples are provided for illustrative purposes only to facilitate a more complete understanding of representative embodiments.

[0329] These examples should not be construed as limiting any of the embodiments described herein, including those relating to methods of treating acquired and hereditary von Willebrand disease.

[0330] Example 1: Purification of mature rVWF on a cation exchanger to separate the cVWF propeptide from the mature rVWF. Example 1 describes the purification of mature rVWF on a cation exchanger (cation exchange (CEX) resin). The rVWF propeptide (rVWF-PP), which remains bound to rVWF after furin maturation, was released using sodium citrate as a chelating agent at neutral pH before loading onto the CEX resin. Most of the rVWF propeptide passed through the cation exchange resin, and the remaining rVWF propeptide was depleted after a washing step. Sodium citrate was used as a buffer component and as a chelating agent.

[0331] In industrial applications, VWF, particularly recombinant VWF (rVWF), is synthesized and expressed together with rFVIII in genetically modified CHO cell lines. The function of the coexpressed rVWF is to stabilize rFVIII during the cell culture process. rVWF is synthesized intracellularly as a pro-form with a high molecular weight propeptide attached to the N-terminus. Upon maturation in the endoplasmic reticulum and Golgi apparatus, rVWF-PP is cleaved by the action of the intracellular protease furin and secreted as a homopolymer of identical subunits consisting of a dimer of the expressed protein. However, maturation is incomplete, resulting in a product containing a mixture of rVWF-PP and mature VWF.

[0332] After the monoclonal antibody step capturing recombinant Factor VIII, the flow-through containing rVWF (also called monoclonal antibody effluent) was loaded onto an anion exchanger (anion exchange (AEX) resin). rVWF was bound to the anion exchanger and matured with furin in the presence of calcium. rVWF was eluted from the anion exchanger with increasing conductivity. The product containing eluate was adjusted to a conductivity of 13.39 mS / cm and pH 7.39 by 1:2 dilution with 60 mM sodium citrate, pH 7.6. The adjusted aqueous dilution was loaded onto a UNOsphere™ S Cation Exchange Media (BioRad, catalog no. 156-0115) cation exchange column with an internal diameter of 15 mm, a bed height of 14.0 cm, and a volume of 24.74 ml at a flow rate of 100 cm / h, followed by washing with 5 CV of 10 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 to remove host cell proteins (HCPs) and rVWF-PP. rVWF was eluted by increasing the conductivity of a linear gradient from 10 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 to 500 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 at a flow rate of 60 cm / h. The main elution peak was split into two portions, allowing the separation of low-molecular-weight and high-molecular-weight rVWF multimers.

[0333] FIG. 1 shows the purification of mature rVWF on a cation exchanger as shown in Example 1.

[0334] FIG. 2 shows a table of the purification results.

[0335] FIG. 3 shows a silver-stained protein gel and Western blot demonstrating the separation of rVWF and rVWF-PP by the method described in Example 1.

[0336] Examples 2 and 3: Optimization of the method described in Example 1 for commercial production of rVWF. Examples 2 and 3 represent the optimization method described in Example 1 for commercial production of rVWF.

[0337] For Examples 2 and 3, an experimental setup for the fermentation of rVWF and rFVIII was established, which was used for a simple purification method to obtain highly pure rVWF for biochemical characterization.

[0338] The capture step was performed by tandem chromatography, combining affinity and anion exchange chromatography in a single process. rFVIII was bound to an anti-FVIII mAb column at temperatures between 2 and 8°C based on immunoaffinity chromatography technology. This step allowed for the separation of rFVIII from rVWF. The flow-through containing rVWF was diluted online with purified water and directly loaded onto an AEX column within the same chromatography system. After a 15°C increase in temperature to 28°C, recombinant furin maturation was performed on the AEX column. The furin-matured rVWF was eluted by stepwise elution with increasing conductivity. A polishing step was also performed. The AEX eluate containing rVWF was diluted with 10 mM sodium citrate buffer, pH 7.6, and loaded onto a UNOsphere™ S Cation Exchange Media (BioRad, catalog no. 156-0115) cation exchange column with an internal diameter of 15 mm, a bed height of 14.0 cm, and a column volume of 25 ± 0.5 ml at a flow rate of 100 cm / h. After a wash step with 10 mM NaCl, 30 mM sodium citrate, 2 mM citric acid, pH 7.6 ± 0.2, rVWF was eluted with a linear gradient of increasing conductivity from 10 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 to 500 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 at a flow rate of 65 cm / h. The main elution peak was collected as the pooled eluate for analytical purposes.

[0339] In the final experimental design, the last 30–40% of the peak was collected to obtain rVWF with the highest specific activity.

[0340] FIG. 4 shows a flow chart of the experimental setup for Examples 2 and 3.

[0341] FIG. 5 shows the chromatogram of Example 2 and the chromatographic scheme used in Examples 2 and 3.

[0342] FIG. 6 shows a table of reagents used in Example 2 and a table of results.

[0343] FIG. 7 shows another chromatogram of Example 2 and a table of the results of Example 3.

[0344] FIG. 8 shows a silver stained protein gel showing the separation of rVWF and rVWF propeptide by the methods of Examples 2 and 3.

[0345] FIG. 9 shows a Western blot demonstrating the separation of rVWF and rVWF propeptide by the methods of Examples 2 and 3.

[0346] Example 4: Commercial production method of rVWF by separating rVWF and rVWF-PP by size exclusion chromatography Example 4 presents a method optimized for commercial production of rVWF by separating rVWF and rVWF propeptide (rVWF-PP) via size exclusion chromatography. The addition of sodium citrate to the SEC running buffer efficiently resolves rVWF and rVWF-PP.

[0347] The post-ultrafiltration UNOsphere™ S eluate containing rVWF was directly loaded onto a series array of two Superose 6 prep grade SEC columns (GE Healthcare, catalog number 28-9913-16), each with an internal diameter of 16 mm and a bed height of 82.0 cm (2 × 41 cm), with a total column volume of approximately 165 ml. The load was applied at a rate of 7 cm / hr. The running buffer was 20 mM HEPES free acid, 150 mM NaCl, 15 mM citric acid dihydrate, pH 7.5 ± 0.2. Size exclusion chromatography was performed using isotonic conditions at a linear flow rate of 12 cm / hr.

[0348] FIG. 10 shows the chromatogram, chromatography scheme, and buffer composition of Example 4.

[0349] FIG. 11 shows a table of the results of Example 4.

[0350] FIG. 12 shows a silver stained protein gel and Western blot demonstrating the separation of rVWF and rVWF propeptide by the method of Example 4.

[0351] Example 5: Optimized method for commercial production of mature rVWF by separation of rVWF and rVWF-PP by size exclusion chromatography. Example 5 illustrates a method for separating rVWF and rVWF-PP by size exclusion chromatography by loading a starting material containing pH-adjusted rVWF onto the size exclusion chromatography.

[0352] The post-ultrafiltration UNOsphere™ S-eluate containing rVWF was adjusted to pH 7.5±0.2 with 1 M glycine pH 9.0 before loading onto the column. This solution was loaded onto a series array of two Superose 6 prep grade SEC columns (GE Healthcare, catalog number 28-9913-16), each with an internal diameter of 16 mm and a bed height of 82.0 cm (2 × 41 cm), with a total column volume of approximately 165 ml. Loading was performed at a flow rate of 7 cm / h. The SEC running buffer contained 20 mM HEPES free acid and 150 mM NaCl, pH 7.5±0.2. Size exclusion chromatography was performed using isotonic conditions at a linear flow rate of 12 cm / h.

[0353] FIG. 13 shows the chromatogram, chromatography scheme, and buffer composition of Example 5.

[0354] FIG. 14 shows a table of the results of Example 5.

[0355] Example 6: CEX method for purifying rVWF from rVWF propeptide on UNOsphere™ S without supplementation with chelating agents Example 6 illustrates a CEX method on ultrafiltered UNOsphere™ S without supplementation with a chelating agent. This method is representative of conventional methods for purifying mature rVWF from rVWF propeptide. The method does not utilize buffers containing chelating agents and / or buffers with a pH above 7.0.

[0356] After the monoclonal antibody step capturing recombinant factor VIII, the flow-through containing rVWF was loaded onto an anion exchanger. rVWF was bound to the anion exchanger and matured with furin in the presence of calcium. rVWF was eluted from the anion exchanger with increasing conductivity. The product-containing eluate was then loaded onto a UNOsphere™ S Cation Exchange Media (BioRad, catalog no. 156-0115) cation exchanger column with an internal diameter of 15 mm, a bed height of 14.2 cm, and a volume of 25.09 ml at a flow rate of 100 cm / h, followed by a 10 CV wash with 10 mM Tris-HCl, 100 mM sodium acetate, 85 mM NaCl, pH 6.5 ± 0.2 to remove HCP and rVWF propeptide. rVWF was eluted in a single step by adding 100 mM sodium acetate, 500 mM NaCl, 100 mM glycine, 3 mM CaCl, pH 7.5±0.2 at a flow rate of 65 cm / h. The main elution peak was collected as the product-containing fraction.

[0357] FIG. 15 shows the chromatogram, chromatography scheme, and buffer composition and conditions of Example 6.

[0358] FIG. 16 shows a table of the results of Example 6.

[0359] Example 7: SEC method for purifying rVWF from rVWF propeptide without prior chelator supplementation or pH increase Example 7 illustrates an SEC method without prior chelating agent supplementation or pH increase. This method is representative of conventional methods for purifying mature rVWF from rVWF propeptide. The SEC method does not include buffers containing chelating agents and / or buffers with a pH of 7.0 or higher used to prepare a starting fraction (material) containing rVWF and residual rVWF propeptide.

[0360] The post-ultrafiltration UNOsphere™ S-eluate containing recombinant VWF was directly loaded onto a series array of two Superose 6 prep grade SEC columns (GE Healthcare, catalog number 28-9913-16), each with an internal diameter of 16 mm and a bed height of 82.0 cm (2 × 41 cm), with a total column volume of approximately 165 ml. The load was applied at a flow rate of 7 cm / h. The running buffer was 20 mM HEPES free acid, 150 mM NaCl, pH 7.5 ± 0.2. Size exclusion chromatography was performed using isotonic conditions at a linear flow rate of 12 cm / h.

[0361] FIG. 17 shows the chromatogram, chromatography scheme, and buffer composition of Example 7.

[0362] FIG. 18 shows a table of the results of Example 7.

[0363] Example 8: Separation of rVWF from rVWF propeptide by anion and cation exchange chromatography. Example 8 illustrates the purification of mature rVWF on a cation exchanger. Starting material was obtained from a current rVWF manufacturing process after the AEX Mustang Q step. The rVWF-containing flow-through from the AEX Mustang Q step was subjected to SD / VI treatment and diluted with a buffer containing a chelating agent to dissociate the rVWF / rVWF propeptide complex. The diluted material was applied to a CEX resin (Unosphere S). Most of the rVWF-PP, host cell proteins (HCPs), and low-molecular-weight rVWF multimers passed through the cation exchange resin. The remaining rVWF-PP was depleted after a washing step. The bound high-molecular-weight rVWF multimers were then eluted by an increase in conductivity caused by sodium ions.

[0364] In industrial applications, VWF, particularly recombinant VWF (rVWF), is synthesized and expressed together with rFVIII in genetically modified CHO cell lines. The function of the coexpressed rVWF is to stabilize rFVIII during the cell culture process. rVWF is synthesized intracellularly as a pro-form with a high molecular weight propeptide attached to its N-terminus. Upon maturation in the endoplasmic reticulum and Golgi apparatus, the propeptide is cleaved by the action of the intracellular protease furin, and the expressed protein is secreted as a homopolymer of identical subunits consisting of a dimer. However, maturation is incomplete, resulting in a product containing a mixture of the propeptide and mature VWF.

[0365] After a monoclonal antibody step capturing recombinant factor VIII, the flow-through containing rVWF was loaded onto a Fractogel TMAE anion exchanger. rVWF bound to the anion exchanger and then matured with furin in the presence of calcium. rVWF eluted from the anion exchanger with increasing conductivity. The TMAE eluate was filtered through a Mustang Q (MUQ) filter unit to remove CHO-DNA and impurities bound to the filter membrane. The loading material for the CEX step was the effluent from the Mustang Q filtration step (MUQ), which inactivates lipid-enveloped viruses by treatment with solvents and detergents. To inactivate viruses, the MUQ effluent was incubated for 1 hour at room temperature with a mixture of two detergents, Triton-X-100 (1%) and polysorbate 80 (0.3%), and the organic solvent tri-n-butyl phosphate (0.3%). The MUQ flow-through containing the product was diluted 1:2 with 60 mM sodium citrate, pH 7.6, to a conductivity of 21.9 mS / cm and a pH of 7.16. High conductivity was selected to ensure removal of rVWF propeptide (rVWF-PP) and low-molecular-weight rVWF multimers, taking advantage of the resin's capacity for the desired high-molecular-weight rVWF multimers. The adjusted dilution was loaded onto a UNOsphere™ S Cation Exchange Media (BioRad, catalog no. 156-0115) cation exchange column with an internal diameter of 10 mm, a bed height of 14.3 cm, and a volume of 11.23 ml at a flow rate of 100 cm / h. After loading, a first wash (re-equilibration) was performed with 5 CV of 10 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 to remove weakly bound HCP and rVWF propeptide.

[0366] A second wash to deplete tightly bound HCP and rVWF propeptide was performed with a step of 40% 500 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 in 10 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 (wash 2).

[0367] Elution was performed in two stages: (1) the first stage included a step of 45% 500 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 in 10 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 (eluate 1, or E1), and (2) the second stage included a linear gradient from 45% to 100% 500 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 in 10 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 (eluate 2, or E2) over 6 column volumes. The elution from wash 2 to the end of the gradient was performed at a flow rate of 65 cm / h.

[0368] FIG. 19 shows the chromatogram, chromatography scheme, and buffer composition of Example 8.

[0369] FIG. 20 shows a table of the results of Example 8.

[0370] FIG. 21 shows a silver stained protein gel showing the separation of rVWF and rVWF propeptide by the method of Example 8.

[0371] Figure 22 shows a Western blot demonstrating the separation of rVWF and rVWF propeptide by the method of Example 8. The 1% agarose gel shows the multimeric pattern of the products.

[0372] FIG. 23 shows a Western blot demonstrating the separation of rVWF and rVWF propeptide by the method of Example 8.

[0373] Example 9: Separation of rVWF from rVWF propeptide by anion and cation exchange chromatography. Example 9 describes the optimized purification of mature rVWF on a cation exchanger. Starting material was obtained from a current r-VWF manufacturing process after the AEX Mustang Q step. The rVWF-containing flow-through from the AEX Mustang Q step was treated with SD / VI and diluted with a buffer containing a chelating agent to dissociate the rVWF / rVWF propeptide complex. The diluted material was loaded onto a CEX resin (Unosphere S). Most of the rVWF-PP, host cell proteins, and low-molecular-weight rVWF multimers pass through the cation exchange resin. The remaining rVWF-PP was depleted after a washing step. The bound high-molecular-weight rVWF multimers were eluted by a gradient of increasing conductivity caused by sodium ions.

[0374] In industrial applications, VWF, particularly recombinant VWF (rVWF), is synthesized and expressed together with rFVIII in genetically modified CHO cell lines. The function of the coexpressed rVWF is to stabilize rFVIII during the cell culture process. rVWF is synthesized intracellularly as a pro-form with a high molecular weight propeptide attached to its N-terminus. Upon maturation in the endoplasmic reticulum and Golgi apparatus, the propeptide is cleaved by the action of the intracellular protease furin, and the expressed protein is secreted as a homopolymer of identical subunits consisting of a dimer. However, maturation is incomplete, resulting in a product containing a mixture of the propeptide and mature VWF.

[0375] After a monoclonal antibody step capturing recombinant factor VIII, the flow-through containing r-VWF was loaded onto a Fractogel TMAE anion exchanger. rVWF was bound to the anion exchanger and matured with furin in the presence of calcium. rVWF eluted from the anion exchanger with increasing conductivity. The TMAE eluate was filtered through a Mustang Q (MUQ) filter unit to remove CHO-DNA and impurities that bound to the filter membrane. The loading material for the CEX step was the wastewater from the Mustang Q filtration step (MUQ), which involves treatment with solvents and detergents to inactivate lipid-enveloped viruses.

[0376] To inactivate the virus, the MUQ wastewater was incubated with a mixture of two surfactants, Triton-X-100 (1%) and polysorbate 80 (0.3%), and the organic solvent tri-n-butyl phosphate (0.3%) at room temperature for 1 hour. The MUQ flow-through containing the product was diluted 1:2 with 60 mM sodium citrate, pH 7.6, to a conductivity of 21.9 mS / cm and a pH of 7.16. A high conductivity was selected to ensure removal of rVWF propeptides and low-molecular-weight rVWF, taking advantage of the resin's capacity for the desired high-molecular-weight rVWF. The adjusted dilution was loaded onto a UNOsphere™ S Cation Exchange Media (BioRad, Cat. No. 156-0115) cation exchange column with an internal diameter of 10 mm, a bed height of 14.3 cm, and a volume of 11.23 ml at a flow rate of 100 cm / h, followed by a first wash (re-equilibration) with 5 CV of 10 mM NaCl, 30 mM sodium citrate, pH 7.6±0.2 to remove weakly bound HCP and rVWF propeptide.

[0377] A second wash (wash 2), which depletes tightly bound HCP and rVWF propeptide, was performed with a step of 36% 500 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 in 10 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 in 5 column volumes.

[0378] Elution was performed with a gradient from 36% 500 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 in 10 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 to 100% 500 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 in 10 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 in 8 column volumes. The eluate corresponding to the desired product included a pool of fractions from >50% 500 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 in the starting 10 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 to 76% 500 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 in 10 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2. Washing and elution were performed at a flow rate of 50 cm / h.

[0379] FIG. 24 shows the chromatogram, chromatography scheme, and buffer composition of Example 9.

[0380] FIG. 25 shows a table of the results of Example 9.

[0381] FIG. 26 shows the product table of Example 9.

[0382] FIG. 27 shows a silver stained protein gel showing the separation of rVWF and rVWF propeptide by the method of Example 9.

[0383] Figure 28 shows a Western blot demonstrating the separation of rVWF and rVWF propeptide by the method of Example 9. The 1% agarose gel shows the multimeric pattern of the products.

[0384] FIG. 29 shows a Western blot demonstrating the separation of rVWF and rVWF propeptide by the method of Example 9.

[0385] The rVWF purification process in the presence of chelating agents and / or elevated pH showed high depletion rates of r-VWF propeptide and host cell proteins. The depletion of r-VWF propeptide on a cation exchanger was based on the fact that rVWF-PP does not bind to a cation exchanger in the presence of chelating agents and / or elevated pH. The depletion of rVWF propeptide on size exclusion chromatography was based on the fact that efficient size separation was achieved in the presence of chelating agents and / or elevated pH.

[0386] FIG. 30 shows the purity of the product-containing fractions obtained by enhanced cation exchange chromatography (CEX) used in Examples 1, 2, 3, 6, 8, and 9.

[0387] FIG. 31 shows the depletion factors of product-related impurities for Examples 1, 2, 3, 6, 8, and 9.

[0388] FIG. 32 shows the purity of the product-containing fractions obtained by enhanced size exclusion chromatography (SEC) used in Examples 4 and 5. FIG. 33 shows the depletion factors of product-related impurities for Examples 4 and 5.

[0389] References U.S. Patent No. 8,058,411; Method for producing mature VWF from VWF pro-peptide. Inventors: Wolfgang Mundt, Artur Mitterer, Meinhard Hasslacher, Christa Mayer.

[0390] U.S. Patent No. 6,465,624; Purification of von Willebrand factor by cation exchange chromatography. Inventors: Bernhard Fischer, Oyvind L. Schonberger, Artur Mitterer, Christian Fiedler, Friedrich Dorner, Johann Eibl.

[0391] Example 10: Separation of rVWF from rVWF propeptide by anion exchange chromatography. This study demonstrates that furin-treated mature VWF / VWF-PP complexes can be dissociated (separated) into mature VWF and VWF-PP using anion exchange chromatography and an elution buffer with a high pH (e.g., pH 8.5) and containing a chelating agent (EDTA). Separation was performed on an anion exchanger (AEX), specifically Fractogel TMAE650(M). A solvent-detergent treatment for virus inactivation was also performed on the column for approximately 1 hour. Details of the chromatography experiments are shown in Figures 34-36.

[0392] FIG. 34 shows the buffer formulations and materials used in the TMAE separation method.

[0393] FIG. 35 shows the loading conditions for furin-treated mature VWF / VWF propeptide complexes.

[0394] Figure 36 details the buffers, conditions, parameters, and flow rates of the chromatography method.

[0395] Figure 37 shows a chromatogram of the dissociation of furin-treated mature VWF / VWF propeptide complexes into mature VWF and VWF-propeptide (VWF-PP), demonstrating the depletion of VWF-PP from fractions containing mature VWF.

[0396] Figure 38 shows another chromatogram of the separation of mature VWF and VWF-propeptide (VWF-PP), demonstrating the depletion of VWF-PP from fractions containing mature VWF.

[0397] Example 11: Improvement of different chromatographic methods for the separation of mature VWF (matVWF) and VWF propeptide (VWF-PP). In the first study, two methods for purifying recombinant mature VWF were compared.

[0398] Figure 39 shows an outline of two methods for isolating mature VWF. For example, in one method, downstream processing steps after obtaining mAb effluent (MABEffl) include a TMAE anion exchange chromatography capture step and on-column maturation (TMC) and a Mustang Q negative anion exchange chromatography step (MUQ), followed by a solvent-detergent treatment (SDT) for viral inactivation, cation exchange chromatography (CAT), ultrafiltration concentration (UFA), size exclusion chromatography (SEC), and dialysis-ultrafiltration concentration (DUF) to produce bulk drug substance (mature VWF). In the other method, downstream processing steps include an improved cation exchange chromatography (CAT) step followed by a dialysis-ultrafiltration (DUF) concentration step to produce bulk drug substance, but do not include SEC.

[0399] Figure 40 shows a table highlighting some of the advantages of the improved cation exchange chromatography method (CAT2.0) described herein and shown in Figure 39. The improved CAT method can remove: host cell impurities by a reduction factor of more than 1000, VWF-PP by a reduction factor of more than 2000, and residual FVIII by a reduction factor of less than 10. The CAT method can be used to separate and pool VWF multimers. Furthermore, this method can replace size exclusion chromatography as a polishing step to isolate the active fraction of VWF and remove remaining host cell-derived impurities and VWF-PP.

[0400] In the second study, the conditions of the SEC process were varied to improve the separation of mature VWF and VWF-PP. In other words, it was determined that the modified SEC buffer could increase the purity of mature VWF by reducing the amount of VWF-PP.

[0401] Figure 41 shows two schematic chromatograms illustrating the separation of r-VWF propeptide using size exclusion chromatography with standard SEC buffer (SQA buffer) or modified SEC buffer (SQC buffer). Figure 42 shows a table highlighting some of the advantages of using SQC buffer. For example, the method using SQC buffer can remove host cell impurities by a reduction factor of more than about 100 and residual FVIII by a reduction factor of less than 10. Surprisingly, this method can remove VWF-PP to impurity levels below 2 μg / 1000 units.

[0402] Thus, this example describes a method for improving the separation of mature VWF from VWF-PP.

[0403] Example 12: Development of an improved CAT (UNO_S) process. The downstream processing of first-generation recombinant von Willebrand factor (rVWF), starting from monoclonal antibody (MAB) flow-through, involves a polishing step by cation exchange chromatography (CAT) on UNO_Sphere S (UNO_S) resin. The UNO_S eluate is then concentrated by ultrafiltration and further processed by size exclusion chromatography (SEC) to separate high- and low-molecular-weight rVWF multimers and remove free rVWF propeptide, a product-related impurity generated during downstream processing. The high-molecular-weight rVWF fraction corresponds to the bulk drug substance (BDS), which is ultimately formulated to obtain the final drug product (FDP).

[0404] In the downstream process of second-generation rVWF, an improved cation exchange chromatography method was proposed to replace the SEC step and separate high- and low-molecular-weight rVWF multimers and rVWF propeptides by an alternative cation exchange (CAT) elution procedure (gradient elution instead of step elution). This example outlines the experimental results of the second-generation rVWF polishing purification step, CAT. Novel process parameters, such as CAT loading pH and conductivity, conductivity and length of the column wash step, and eluate pooling criteria, were investigated on a small scale to obtain a scalable and robust downstream unit operation.

[0405] 1. Purpose The downstream processing of first-generation rVWF (VONVENDI®) begins with a TMAE Sepharose capture step (TMC step) using ADVATE® MAB flow-through as a feed, followed by a Mustang Q filtration step to remove CHO host cell DNA. A solvent / detergent (S / D) step is then performed to inactivate latent lipid-enveloped viruses, followed by a polishing step (CAT step) on a weak cation exchanger, UNO_SphereS (UNO_S) resin. The CAT step aims to remove S / D chemicals introduced during the virus inactivation step. The UNO_S eluate is then concentrated by ultrafiltration and further processed by size exclusion chromatography (SEC) to separate high- and low-molecular-weight rVWF multimers and remove free rVWF propeptide, a product-related impurity generated during downstream processing. The high-molecular-weight rVWF subfraction corresponds to the BDS that is ultimately formulated to obtain FDP.

[0406] For downstream processing of second-generation rVWF, it was proposed to omit the SEC step and replace it with a modified cation exchange chromatography method.

[0407] In a series of five experiments, separation of high-molecular-weight rVWF multimers from low-molecular-weight rVWF multimers and removal of rVWF propeptide were achieved by a gradient CAT elution procedure. The novel gradient elution mode could replace the stepwise elution procedure applied in the first-generation downstream process. In this example, five experiments for the second-generation rVWF polishing purification step, CAT, are outlined in detail. All experiments were performed according to the research protocol described herein.

[0408] 2. Introduction and Background The current report describes the development of a second-generation (Gen2) T process by combining two VWF downstream unit operations, CAT and SEC, currently applied in the first-generation (Gen1) procedure. A series of experiments investigated process parameters identified in the risk assessment and considered critical for the performance of the chromatographic step, CAT. The current study was based on a scale-down model from the current rVWF manufacturing process. This process was established at Orth for the manufacture of clinical Phase III material and was transferred to manufacturing (MFG) scale for commercial production (Figure 43A). To facilitate understanding of the changes introduced in the CAT unit operation step described in this report, a brief process description of the currently used first-generation rVWF downstream unit operations, S / D, CAT, and SEC, is provided below.

[0409] When used in the Gen1 process, the rVWF polishing step, CAT, is a chromatographic cation exchange process on UNO_Sphere S, a macroporous acrylamide-based medium with "strong" sulfonic acid cation exchange ligands. The loading material for the polishing step is the wastewater from the anion exchange filtration step, MUQ, which is treated with solvents and detergents to inactivate lipid-enveloped viruses. To inactivate the virus, the MUQ wastewater is incubated for 1 hour at room temperature with a mixture of two detergents, Triton-X-100 (1%) and polysorbate 80 (0.3%), and the organic solvent tri-n-butyl phosphate (0.3%). Prior to processing, the product solution is filtered through a 0.2 μm membrane filter to remove any particles that may be present. After virus inactivation, the product solution is diluted with approximately 1 volume of water to reduce the concentration of the S / D reagents and adjust the conductivity for the CAT column loading step. The pH is not adjusted. The CAT chromatography step is primarily intended to remove S / D reagents and further reduce process-related impurities, including medium components such as soy peptone, rfurin, rFVIII polypeptide, and other impurities such as CHO-derived proteins and DNA. Following unit operation step CAT, the resulting product fraction (CAT-E) is further processed by size-exclusion chromatography (SEC) on Superose 6 resin. The loading material for step SEC is the eluate pool from step CAT on UNO_Spheres. Because the loading capacity of the SEC column is limited to achieve reasonable resolution, the CAT eluate pool is concentrated approximately 15-fold by ultrafiltration (step UFA) using a cellulose-based membrane cassette with a 30 kDa cutoff. At a clinical Phase III production scale, the ultrafiltration concentration (UFA) concentrate is divided into two fractions, which are processed separately on the SEC column. This measure was implemented to keep the SEC column volume and column diameter low. The conductivity and pH of the buffer matrix as well as the loading material correspond to the CAT eluate pool and are not adjusted after the UFA concentration step before loading onto the SEC column.The purpose of step SEC is the final impurity removal of CHO host cell proteins and serves as the primary removal step for rVWF propeptide, a product-associated impurity generated in the initial capture step on TMAE Sepharose (TMC step). Additionally, step SEC separates rVWF multimers based on size, enabling a pooling scheme to enrich for high-molecular-weight rVWF multimers that contribute to the ristocetin cofactor activity of the product.

[0410] This report describes the replacement of a current unit operation process run at MFG (Figure 43A) scale with an improved CAT (UNO_S) process (Figure 43B). The CAT process improvements were investigated at a small scale. The UDF (concentration / dialysis) process following the CAT process may also need to be optimized.

[0411] 3. Materials and Methods The materials and methods, as well as the sampling plan, are described herein.

[0412] 3.1 rVWF loading material Frozen MUQ-E product was used for all experiments. The material was stored frozen in 130 mL aliquots at ≤-60°C and thawed overnight between +2 and +8°C as needed. Once the MUQ-eluate was thawed, the S / D reagent was added, and the mixture was filtered through a 0.2 μm Pall filter (KA02EAVP2S®). The filtered material was then incubated at room temperature (approximately +25°C) for 60 minutes under moderate agitation to inactivate / dissolve latent lipid-enveloped viruses. The S / D reaction was terminated by a 1:2 dilution with 60 mM sodium citrate buffer, pH 7.5. The diluted material was used as feed for the following CAT step.

[0413] 3.2 Chromatography Hardware The experiments described in the current report used a small-scale chromatography system, AKTA pure25 (GE Healthcare). The system was equipped with probes for online monitoring of UV absorbance, conductivity, pressure, temperature, and pH, along with electronic recordings. The system was controlled by software running Unicorn 7.0. All experiments were performed at ambient room temperature.

[0414] The AKTA system tubing was PEEK, unlike the larger scale Millipore process system, which uses stainless steel tubing. All hardware components are certified R&D equipment.

[0415] The laboratory-scale columns used in all five experiments were equipped with a 10 μm PP frit; the particle size of the UNO_Sphere S resin was approximately 80 μm in diameter. For larger scales, stainless steel frits with a 20 μm mesh size were used. All columns were certified and designed for research and development purposes.

[0416] A hardware comparison between the current GEN1 MGF instrument at NE and the small-scale GEN2 instrument used in the current study is shown in Figure 44 .

[0417] 3.3 Buffer Buffers used for small-scale purification runs were made in the laboratory area or received from the manufacturing area. Certified chemicals were used for buffer preparation, which were also used to produce buffers for pilot-scale clinical production. Buffers were 0.2 μm filtered before use and stored in bags or glass bottles at room temperature. A description of the buffer compositions is shown in Figure 48.

[0418] 3.4 Analysis method rVWF biochemical characterization, potency, and impurity assays performed include those analyzing VWF:RistoCo activity, VWF antigen, VWF propeptide antigen content, FVIII activity chromogenic assay, UV absorption characteristics (280 nm, 254 nm), polypeptide patterns (e.g., degradation, multimerization pattern), and CHO HCP content. In some cases, other analytical tests can be performed to measure, for example, but not limited to, pro-VWF antigen content, FVIII antigen content, furin activity, furin antigen, total protein (BCA), free sulfhydryls, CHO BIP WB, CHO DNA, mouse monoclonal antibodies, soy peptone, Triton X-100, polysorbate 80, tri-n-butyl phosphate, dynamic light scattering (DLS) (hydrodynamic radius), sialic acid, n-glycan content, VWF collagen binding, and VWF oxidation.

[0419] 4 Changes in CAT Process rVWF 2nd Generation (GEN2) The following parameters were considered to replace the SEC step in the second-generation rVWF downstream process. Most of the changes introduced were based on R&D feasibility studies. The chromatography resin type (UNO_Sphere S resin by BioRad) and the composition of the added buffer (but not pH) were not changed. The second-generation CAT process included: changes in S / D treatment, loading concentration and flow rate, and washing and elution steps.

[0420] 4.1 S / D processing S / D inactivation was stopped by diluting the virus inactivating material 1:2 with 60 mM sodium citrate buffer, and this dilution allowed the CAT feed to reach a preferred pH of 7.5–8.0 (pH test range 6.0–9.0) and a pH of 10–30 mS / cm at +25°C. 2 Preferred conductivity (conductivity test range 5-40 mS / cm at +25°C) 2The S / D treatment was performed similarly to the GEN1 process, except that the pH was set to 8.9-9.2. In the GEN1 rVWF CAT step, the CAT load was set to pH 8.9-9.2. In the GEN2 setup, the conductivity and pH were set to minimize binding of CHO-HCP, CHO-DNA, and rVWF propeptide to the matrix. Similarly, low-molecular-weight (LMW) rVWF molecules were prevented from binding to the column matrix, while only high-molecular-weight (HMW) rVWF molecules were preferably captured. One of the goals of this study was to increase the conductivity during the loading step and deplete as much LMW rVWF, CHO-HCP, CHO-DNA, and rVWF propeptide as possible from the feed.

[0421] 4.2 Loading concentration and flow rate The loading concentration (RU rVWF / mL resin) was increased during the study to allow for more product to be loaded without increasing the column volume. In contrast to the commonly applied loading concentrations of 60-140 RU / mL resin at manufacturing scale (MFG), 90-270 IU / mL resin was loaded in the current small-scale study. The flow rates for equilibration, loading, and re-equilibration in the second-generation CAT procedure were the same as those in the first-generation process (100 cm / hr). The flow rates for wash and elution were varied as shown in Figure 45.

[0422] 4.3 Loading concentration and flow rate The wash steps preceding the elution step were modified to optimize the removal of process- and product-related impurities. The step elution applied in the first-generation process was changed to gradient elution. The length of the gradient was explored during the study. The modification of the elution procedure was based on the observation that low molecular weight rVWF molecules are eluted in early gradient fractions and high molecular weight rVWF molecules are eluted in later gradient fractions (see, e.g., US 6,465,624).

[0423] 5. Comparison of CAT Gen1 and Gen2 Processes In both procedures, the CAT process involves the following steps: column activation (loading of anionic ligands with cationic counterion sodium) and equilibration (preparing the column for loading in terms of stable pH and conductivity (monitored at the column outlet)), followed by product loading of the S / D treated and diluted MUQ eluate.

[0424] During loading at the MFG scale, the eluate was filtered online through a 0.2 μm filter to protect the column from particulate matter that may have formed during the S / D process. This step was omitted in the small-scale process. After pumping the product-containing solution onto the column, loading was completed, and loosely bound impurities were removed by applying a wash step to remove the low-molecular-weight S / D reagents that had been pumped onto the column. The pH and conductivity of the wash step corresponded to the parameters of the equilibration and loading steps. After washing, bound proteins were eluted from the column by applying a stepwise elution using an elution buffer with a high conductivity and counterion concentration. A product pool of ≤3.6 CV was collected.

[0425] In the small-scale Gen2 process, an alternative gradient elution procedure was used to remove rVWF propeptides, low-molecular-weight rVWF molecules, and high-molecular-weight molecule...

Claims

1. A method for obtaining a composition comprising highly purified mature recombinant rVWF (mat-rVWF) depleted of propeptides, comprising the steps of: (a) providing a furin-treated solution containing mat-rVWF / rVWF-PP complexes, mat-rVWF, and rVWF propeptide (rVWF-PP); (b) after step (a), dissociating the mat-rVWF / rVWF-PP complex in the solution of (a) into mat-rVWF and rVWF-PP by contacting the solution with a buffer comprising at least one chelating agent and a pH of at least 7, wherein the dissociation occurs by disruption of non-covalently associated mat-rVWF and rVWF-PP; and (c) after step (b), recovering the mat-rVWF to obtain a highly purified mat-rVWF composition containing at least 95% mature rVWF and less than 5% rVWF-PP, wherein the recovering step comprises one or more protein separation methods selected from the group consisting of ion exchange chromatography (IEC), size exclusion chromatography (SEC), physical size separation by membrane technology, and affinity chromatography.

2. 2. The method of claim 1, wherein the highly pure mat-rVWF composition comprises at least 96% mat-rVWF and less than 4% rVWF-PP.

3. 3. The method of claim 1 or 2, wherein the solution is selected from the group consisting of cell culture medium, antibody column flow-through solution, and buffer solution.

4. The method according to any one of claims 1 to 3, wherein the solution is an antibody column flow-through solution.

5. The method of any one of claims 1 to 4, wherein the at least one chelating agent is a divalent cation chelating agent.

6. 6. The method of claim 5, wherein the divalent cation chelator is selected from the group consisting of EDTA, EGTA, CDTA, and citrate.

7. 7. The method of any one of claims 1 to 6, wherein the pH is increased to at least 7.

1.

8. 8. The method of any one of claims 1 to 7, wherein the pH is increased to at least 7.

2.

9. 9. The method of any one of claims 1 to 8, wherein the pH is increased to at least 7.

6.

10. 10. The method of any one of claims 1 to 9, wherein the pH is increased by the addition of a basic amino acid, Tris, NaOH, Tricine, or ethanolamine.

11. 11. The method of any one of claims 1 to 10, wherein the recovering in step (c) of claim 1 comprises one or more protein separation methods.

12. 12. The method of claim 11, wherein the protein separation method is size exclusion chromatography (SEC).

13. 12. The method of claim 11, wherein the one or more protein separation methods is ion exchange chromatography (IEC).

14. 14. The method of claim 13, wherein the ion exchange chromatography (IEC) is cation exchange chromatography.

15. 14. The method of claim 13, wherein the ion exchange chromatography (IEC) is a combination of anion exchange chromatography and cation exchange chromatography.

16. 12. The method of claim 11, wherein the one or more protein separation methods comprise a buffer system, the buffer system comprising one or more buffers.

17. 17. The method of claim 16, wherein the one or more buffers comprise the one or more chelating agents.

18. 17. The method of claim 16, wherein the one or more buffers exhibit a pH of at least 7.

19. 12. The method of claim 11, wherein the one or more protein separation methods comprise a buffer system, and the buffer system comprises one or more loading buffers.

20. 20. The method of claim 19, wherein the one or more loading buffers comprise the one or more chelating agents.

21. 21. The method of claim 19 or 20, wherein the one or more loading buffers exhibit a pH of at least 7.

22. 12. The method of claim 11, wherein the one or more protein separation methods comprise a buffer system, the buffer system comprising one or more loading buffers, wash buffers, and / or elution buffers.

23. 23. The method of claim 22, wherein the one or more loading buffers, wash buffers, and / or elution buffers comprise the one or more chelating agents.

24. 24. The method of claim 22 or 23, wherein the one or more loading, washing and / or elution buffers exhibit a pH of at least 7.

25. 25. The method according to any one of claims 22 to 24, wherein the one or more loading buffers, washing buffers and / or elution buffers comprise the one or more chelating agents and exhibit a pH of at least 7.

26. 26. The method of any one of claims 16 to 25, wherein one or more buffers of the buffer system are selected from the group consisting of glycine HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TrisHCl (tris(hydroxymethyl)-aminomethane), histidine, imidazole, acetate citrate, and MES (2-(N-morpholino)ethanesulfonic acid).

27. 27. The method of any one of claims 16 to 26, wherein the buffer further comprises one or more monovalent cations.

28. 28. The method of claim 27, wherein the one or more monovalent cations are selected from the group consisting of Na+, K+, Li+, and Cs+.

29. 28. The method of claim 27, wherein the monovalent cation is Na+.

30. 30. The method of any one of claims 16 to 29, wherein the buffer further comprises one or more monovalent, divalent and / or trivalent anions.

31. The one or more monovalent, divalent and / or trivalent anions are Cl － , acetic acid － , S.O. ４ ２－ ,Br － , and citric acid ３－ 31. The method of claim 30, selected from the group consisting of:

32. 32. The method of any one of claims 16 to 31, wherein the buffer system comprises at least one buffer solution exhibiting a conductivity of ≥ 0.5 mS / cm at 25°C.

33. 33. The method of any one of claims 16 to 32, wherein the buffer system comprises at least one buffer solution exhibiting a conductivity of 15.0±0.2 mS / cm at 25°C.

34. 34. The method of any one of claims 16 to 33, wherein the buffer further comprises one or more non-ionic detergents.

35. 35. The method of claim 34, wherein the non-ionic surfactant is selected from the group consisting of Triton X-100, Tween 80, and Tween 20.

36. 36. The method of any one of claims 16 to 35, wherein the buffer further comprises one or more additional substances selected from the group consisting of non-reducing sugars, sugar alcohols, and polyols.

37. The method of any one of claims 1 to 36, wherein the highly pure mat-rVWF composition has a host cell (HC) impurity level of ≦2.0%.

38. The method of any one of claims 1 to 37, wherein the highly pure mat-rVWF composition has a host cell (HC) impurity level of ≦0.6%.

39. The method according to any one of claims 1 to 38, wherein the solution comprising mat-rVWF / rVWF-PP complexes, mat-rVWF and rVWF-PP is obtained from a capture step of rVWF.

40. 40. The method of any one of claims 1 to 39, wherein the solution comprising mat-rVWF / rVWF-PP complexes, mat-rVWF, and rVWF-PP is derived from a process comprising a FVIII immunoaffinity step and an anion exchange chromatography step.

41. A method for obtaining a composition comprising highly purified mature recombinant rVWF depleted of propeptides (highly purified mat-rVWF), comprising the steps of: (a) loading a furin-treated solution containing pro-rVWF, mat-rVWF / rVWF-PP complex, mat-rVWF, and / or rVWF propeptide (rVWF-PP) onto an anion exchange column, and allowing the pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF to bind to the anion exchange column; (b) washing the anion exchange column of (a) containing the bound pro-rVWF, mat-rVWF / rVWF-PP complexes, and mat-rVWF with one or more wash buffers; (c) treating the column of (b) containing the bound pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF with additional furin, wherein the furin cleaves the pro-rVWF into mat-rVWF and rVWF-PP; (d) eluting the bound pro-rVWF, mat-rVWF / rVWF-PP complex, and mat-rVWF from the column of (c) with an elution buffer, wherein the elution buffer induces dissociation of the rVWF-PP from mat-rVWF non-covalently associated with the rVWF-PP, and the dissociation is (i) adding at least one chelating agent to said elution buffer; and (ii) increasing the pH of the elution buffer to at least pH 7. inducing by (e) separating and recovering the mat-rVWF from the rVWF-PP to obtain a highly purified mat-rVWF composition, wherein the highly purified mat-rVWF composition contains at least 95% mature rVWF and less than 5% rVWF-PP.

42. 42. The method of claim 41, wherein (a) and (b) are carried out simultaneously in a single step.

43. 43. The method of claim 41 or 42, wherein the solution of (a) comprises flow-through from a monoclonal antibody column, and the monoclonal antibody is a FVIII monoclonal antibody.

44. 44. The method of any one of claims 41 to 43, wherein the solution in (a) is selected from the group consisting of cell culture medium, antibody column flow-through solution, and buffer solution.

45. 45. The method of any one of claims 41 to 44, wherein the at least one chelating agent is a divalent cation chelating agent.

46. 46. ​​The method of claim 45, wherein the divalent cation chelator is selected from the group consisting of EDTA, EGTA, CDTA, and citrate.

47. 47. The method of any one of claims 41 to 46, wherein the pH is increased to at least 7.

1.

48. 48. The method of any one of claims 41 to 47, wherein the pH is increased to at least 7.

2.

49. 49. The method of any one of claims 41 to 48, wherein the pH is increased to at least 7.

6.

50. 50. The method of any one of claims 41 to 49, wherein the pH is increased by the addition of a basic amino acid.

51. 50. The method of any one of claims 41 to 49, wherein the one or more wash buffers in (b) comprise the one or more chelating agents.

52. 50. The method of any one of claims 41 to 49, wherein the one or more wash buffers of (b) exhibit a pH of at least 7.

53. 50. The method of any one of claims 41 to 49, wherein the one or more wash buffers of (b) comprise the one or more chelating agents and exhibit a pH of at least 7.

54. 50. The method of any one of claims 41 to 49, further comprising a viral inactivation step, said viral inactivation being carried out before, after or simultaneously with the washing step and / or the elution step but before the step of recovering.

55. 55. The method of claim 54, wherein the viral inactivation treatment inactivates lipid-enveloped viruses.

56. 56. The method of claim 54 or 55, wherein the virus inactivation treatment is an S / D treatment.

57. 57. The method of any one of claims 41-56, wherein the one or more wash buffers and the elution buffer comprise a buffer selected from the group consisting of glycine, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TrisHCl (tris(hydroxymethyl)-aminomethane), histidine, imidazole, acetate citrate, and MES (2-(N-morpholino)ethanesulfonic acid).

58. 58. The method of any one of claims 41 to 57, wherein the one or more wash buffers and the elution buffer further comprise one or more monovalent cations.

59. 59. The method of claim 58, wherein the one or more monovalent cations are selected from the group consisting of Na+, K+, Li+, and Cs+.

60. 60. The method of claim 59, wherein the monovalent cation is Na+.

61. 61. The method of any one of claims 41 to 60, wherein the one or more wash buffers and the elution buffer further comprise one or more monovalent, divalent, and / or trivalent anions.

62. The one or more monovalent, divalent and / or trivalent anions are Cl － , acetic acid － , S.O. ４ ２－ ,Br － , and citric acid ３－ 62. The method of claim 61 , selected from the group consisting of:

63. 63. The method of any one of claims 41 to 62, wherein the one or more wash buffers and the elution buffer comprise at least one buffer exhibiting a conductivity of ≥ 0.5 mS / cm at 25°C.

64. 64. The method of any one of claims 41 to 63, wherein the one or more wash buffers and the elution buffer comprise at least one buffer exhibiting a conductivity of 15.0±0.2 mS / cm at 25°C.

65. 65. The method of any one of claims 41 to 64, wherein the one or more wash buffers and the elution buffer further comprise one or more non-ionic detergents.

66. 66. The method of claim 65, wherein the non-ionic surfactant is selected from the group consisting of Triton X-100, Tween 80, and Tween 20.

67. 67. The method of any one of claims 41 to 66, wherein the one or more wash buffers and the elution buffer further comprise one or more additional substances selected from the group consisting of non-reducing sugars, sugar alcohols, and polyols.

68. 68. The method of any one of claims 41 to 67, wherein the highly pure mat-rVWF composition has a host cell (HC) impurity level of ≦2.0%.

69. The method of any one of claims 41 to 68, wherein the highly pure mat-rVWF composition has a host cell (HC) impurity level of ≦0.6%.

70. The method according to any one of claims 1 to 69, wherein the highly purified mat-rVWF composition is used for the manufacture of a pharmaceutical composition.

71. The method of any one of claims 1 to 70, wherein the highly pure mat-rVWF composition is lyophilized after purification.

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