EA045553B1 - SEPARATION OF VWF AND VWF PROPEPTIDE BY CHROMATOGRAPHIC METHODS - Google Patents

Description

Cross reference to related applications

The invention claims benefit from US Provisional Application No. 62/646,109, filed March 21, 2018, which is incorporated herein by reference in its entirety.

Technical field

This invention relates to methods for separating mature von Willebrand factor (VWF) from von Willebrand factor propeptide (VWF-PP).

BACKGROUND OF THE INVENTION

During protein maturation within a cell, the maturing protein undergoes post-translational modifications. These modifications include acetylation, methylation, glycosylation, and proteolytic cleavage, among others. These modifications are in many cases essential to the function and activity of proteins and can also affect the efficiency of proteins, particularly enzymes.

Proproteins or protein precursors are inactive proteins that are converted to the active form by one or more of these post-translational modifications, in particular by cleavage of a propeptide from the proprotein.

The active form of these proteins may be useful therapeutic and/or diagnostic proteins. However, active proteins are usually available in very small quantities in living organisms. Essentially, active proteins are produced recombinantly from their proteins, which are preferably activated in vitro by contacting them with recombinant activation enzymes (eg, proteases).

Von Willebrand factor (VWF) is a glycoprotein that circulates in the plasma as a series of multimers ranging in size from about 500 to 20,000 kDa. Full-length VWF cDNA was cloned; The propolypeptide corresponds to amino acid residues 23 to 764 of the full-length prepro-VWF (Eikenboom et al. (1995) Haemophilia, 1, 77-90). Multimeric forms of VWF consist of 250 kDa polypeptide subunits linked together by disulfide bonds. VWF mediates the initial adhesion of platelets to the subendothelium of the damaged vessel wall, with larger multimers exhibiting increased hemostatic activity. Multimerized VWF binds to the platelet surface glycoprotein Gp1ba through an interaction in the A1 domain of VWF, facilitating platelet adhesion. Other sites on the VWF mediate binding to the blood vessel wall. Thus, VWF forms a bridge between platelets and the vessel wall, which is essential for platelet adhesion and primary hemostasis under high shear stress conditions. Typically, endothelial cells secrete large polymeric forms of VWF, and those forms of VWF that have lower molecular weight arise from proteolytic cleavage. Multimers of exceptionally large molecular weights are stored in Weibel-Pallas bodies of endothelial cells and are released upon stimulation with agonists such as thrombin and histamine.

At the industrial level, VWF, particularly recombinant VWF (rVWF), is synthesized and expressed together with rFVIII in genetically engineered cell lines, such as the engineered CHO cell line. The function of coexpressed rVWF is to stabilize rFVIII during cell culture. rVWF is synthesized in the cell as a VWF pre-propeptide (prepro-VWF), containing a large propeptide (VWF-PP) attached to the N-terminus of the mature VWF subunit (matVWF). After maturation in the endoplasmic reticulum and Golgi apparatus, VWF-PP is cleaved by the cellular protease furin and secreted as a homopolymer of identical subunits, consisting of dimers of the expressed protein. In some cases, furin cleavage provides a heterodimeric complex containing mature VWF non-covalently associated with the VWF propeptide.

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; Preininger A. et al. (1999) Cytotechnology 30:1 -15). Furin belongs to the family of proprotein convertases and is Ca ²⁺ dependent. This enzyme specifically cleaves the C-terminal peptide bond of arginine in a specific sequence containing arginine at positions -1 and -4. This sequence can be found in many human proteins, indicating that furin plays a major role in the maturation of a number of human propeptide proteins. The furin used in the method of this invention is preferably of recombinant origin. Recombinantly produced proteases are preferably used since they can be produced in large quantities. In some embodiments, furin is obtained from the crude supernatant of a culture of a cell line secreting said protease or cell extract.

Current conventional methods produce mature VWF either by incubating the VWF pre-pro-peptide with proteases in the liquid phase, whereby the maturation itself (e.g. cleavage of the pro-peptide from the pro-protein) occurs in an unbound state in free solution, or as described in e.g. WO 2000/049047, by immobilizing the protease on a solid support which is contacted and incubated with a preparation containing VWF-PP (see, for example, WO 2000/049047). VWF is synthesized by endothelial cells and megakaryocytes as the VWF pre-propeptide (preproVWF), which consists largely of repeating domains. After cleavage of the signal peptide, prepro-VWF dimerizes through disulfide bonds at the carboxy terminus in the endoplasmic reticulum. Additional disulfide bonds are formed near the amino terminus of the subunits to form multimers in the Golgi complex. Assembly into the multimer is followed by proteolytic cleavage of the VWF propeptide by the propeptide-processing protease, furin. After cleavage, the VWF propeptide remains non-covalently associated with the VWF multimer to form the mature VWF/VWF-PP complex. Upon stimulation, the complex is 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 digestion and purification steps. However, there remains a need in the art for the production of highly pure, therapeutically effective multimeric mature VWF (mat-rVWF) preparations, and the present invention addresses this need by providing methods for producing high purity mat-rVWF preparations, where the method includes: for example, after furin maturation, adding chelating agent and/or increasing the pH to at least pH 7 during the purification process to facilitate separation of the VWF propeptide from matrVWF.

Summary of the invention

The present invention provides a method for producing a composition containing highly purified mature propeptide-deprived recombinant rVWF (mat-rVWF), the method comprising the steps of:

providing a solution containing the mat-rVWF/rVWF-PP complex, mat-rVWF and rVWF propeptide (rVWF-PP);

inducing the dissociation of the specified mat-rVWF/ rVWF-PP complex in the specified solution onto a) onto mat-rVWF and rVWF-PP, wherein said dissociation occurs by destruction of non-covalently bound mat-rVWF and rVWF-PP, while said dissociation is induced by:

adding at least one chelating agent, or increasing the pH to a pH of at least 7; and collecting said mat-rVWF to obtain a highly purified mat-rVWF composition, wherein said highly purified mat-rVWF composition contains at least 95% mature rVWF and less than 5% rVWF-PP.

In some embodiments, the highly purified mat-rVWF composition contains at least 96% mat-rVWF and less than 4% rVWF-PP, at least 97% mat-rVWF and less than 3% rVWFPP, at least 98% mat-rVWF and less 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.

In some embodiments, the solution is selected from the group consisting of cell culture medium, antibody column flow-through solution, and a buffered solution.

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

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

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 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 from 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 adding basic amino acids, Tris, NaOH, tricine, or ethanolamine.

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

In some embodiments, the one or more protein separation methods include a buffer system, wherein the buffer system contains one or more buffers. In some embodiments, said one or more buffers include wash buffers, wherein said one or more wash buffers include one, two, three, four and/or

- 2 045553 five wash buffers, wherein when said one or more buffers include five wash buffers, the first, second, third and/or fifth wash buffers have a higher pH than the fourth wash buffer, and when said one or more buffers include four wash buffers buffers, the first, second and/or fourth wash buffers have a pH greater than that of the third wash buffer. In some embodiments, the method further includes a virus inactivation treatment step after the first wash buffer, and optionally, the pH of the virus inactivation treatment step is greater than the pH of said third and/or fourth wash buffer. In some embodiments, one or more buffers contain said one or more chelating agents. In some embodiments, the one or more buffers have a pH of at least 7.

In some embodiments, said one or more protein separation methods include a buffer system, wherein said buffer system includes one or more loading buffers. In some embodiments, one or more loading buffers contain said one or more chelating agents. In some embodiments, one or more loading buffers have a pH of at least 7.

In some embodiments, said one or more protein separation methods include a buffer system, wherein said buffer system includes one or more loading, wash and/or elution buffers. In some embodiments, one or more loading, wash and/or elution buffers contain said one or more chelating agents. In some embodiments, one or more loading, wash and/or elution buffers have a pH of at least 7. In some embodiments, one or more loading, wash and/or elution buffers contain said one or more chelating agents and have a pH of at least 7.

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

In some embodiments, the buffer further contains 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 buffer further contains one or more monovalent, divalent and/or trivalent anions. In some embodiments, one or more monovalent, divalent and/or trivalent anions is selected from the group consisting of Cl, acetate, SO ₄ ^2- , ^Br- and citrate.

In some embodiments, the buffer system contains at least one buffer having a conductivity of >0.5 mS/cm at 25°C. In some embodiments, the buffer system contains at least one buffer having a conductivity of 15.0 ± 0.2 mS/cm at 25°C.

In some embodiments, the buffer further contains one or more non-ionic detergents. In some embodiments, the nonionic detergent is selected from the group consisting of Triton X100, Tween 80, and Tween 20.

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

In some embodiments, the highly purified mat-rVWF composition contains a host cell impurity (HC) level of <2.0%. In some embodiments, the highly purified mat-rVWF composition contains a host cell impurity (HC) level of <0.6%.

In some embodiments, a solution containing a complex of mat-rVWF/rVWF-PP, matrVWF and rVWF-PP is prepared in a capture step for rVWF.

In some embodiments, a solution comprising a mat-rVWF/rVWF-PP complex, matrVWF and rVWF-PP is prepared by a process including an FVIII immunoaffinity step and an anion exchange chromatography step.

The present invention also provides a method for producing a composition containing highly purified mature propeptide-deprived recombinant rVWF (highly purified mat-rVWF), said method comprising the steps of:

loading a solution containing pro-rVWF, mat-rVWF/rVWF-PP complex, mat-rVWF and/or rVWF propeptide (rVWF-PP) onto an anion exchange column, said pro-rVWF, matrVWF/rVWF-PP complex and mat -rVWF are associated with the specified anion exchange column;

washing said anion exchange column in a) containing said bound prorVWF, mat-rVWF/rVWF-PP complex and mat-rVWF, with one or more wash buffers;

processing of the indicated column in b), containing bound pro-rVWF, complex mat-rVWF/rVWF- 3 045553

PP and mat-rVWF, furin, wherein said furin cleaves said pro-rVWF into mat-rVWF and rVWF-PP;

eluting said bound pro-rVWF, mat-rVWF/rVWF-PP complex and mat-rVWF from the column in c) with an elution buffer, wherein said elution buffer induces dissociation of said rVWF-PP from non-covalently bound mat-rVWF with said rVWF-PP, and wherein said dissociation is induced by:

adding at least one chelating agent to said elution buffer, or increasing the pH of said elution buffer to a pH of at least 7; and collecting said mat-rVWF separately from said rVWF-PP to obtain a highly purified mat-rVWF composition, wherein said highly purified mat-rVWF composition contains at least 95% mature rVWF and less than 5% rVWF-PP.

In some embodiments, a) and b) are performed simultaneously in the same step.

In some embodiments, said one or more buffers include wash buffers, wherein said one or more wash buffers include one, two, three, four and/or five wash buffers, wherein said one or more buffers include five wash buffers, the first, the second, third and/or fifth wash buffers have a higher pH than the fourth wash buffer, and when the one or more buffers include four wash buffers, the first, second and/or fourth wash buffers have a pH greater than the third wash buffer. In some embodiments, the method further includes a virus inactivation treatment step after the first wash buffer, and optionally, the pH of the virus inactivation treatment step is greater than the pH of said third and/or fourth wash buffer.

In some embodiments, the solution in a) includes a flow-through solution from a monoclonal antibody column, wherein said monoclonal antibody is a FVIII monoclonal antibody.

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

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 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 from 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 adding essential amino acids. In some embodiments, the one or more wash buffers at b) contain said one or more chelating agents. In some embodiments, the one or more wash buffers at b) have a pH of at least 7. In some embodiments, the one or more wash buffers at b) contain said one or more chelating agents and have a pH of at least 7.

In some embodiments, the method further includes a viral inactivation step, wherein said viral inactivation occurs before, after, or simultaneously with the washing step and/or the elution step, but before the collection step. In some embodiments, the viral inactivation treatment inactivates lipid enveloped viruses. In some embodiments, the virus inactivation treatment is a solvent and detergent (S/D) treatment.

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, citrate acetate , MES and 2-(Nmorpholino)ethanesulfonic acid.

In some embodiments, the one or more buffers further contain 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 contain one or more monovalent, divalent and/or trivalent anions. In some embodiments, one or more monovalent, divalent and/or trivalent anions is selected from the group consisting of Cl ^- , acetate ^- , SO ₄ ^{2 -} , Br ^- and citrate ^{3 -} .

In some embodiments, the one or more buffers comprise at least one buffer having a conductivity of >0.5 mS/cm at 25°C. In some embodiments, the one or more buffers comprise at least one buffer having a conductivity of 15.0 ± 0.2 mS/cm at

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25°C.

In some embodiments, the one or more buffers further contain one or more non-ionic detergents. In some embodiments, the nonionic detergent is selected from the group consisting of Triton X100, Tween 80, and Tween 20.

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 highly purified mat-rVWF composition contains a host cell impurity (HC) level of <2.0%. In some embodiments, the highly purified mat-rVWF composition contains a host cell impurity (HC) level of <0.6%.

In some embodiments, the highly purified mat-rVWF composition is used to produce a pharmaceutical composition.

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

Other objects, advantages and embodiments of the invention will become apparent from the following detailed description.

Brief description of graphic materials

In fig. Figure 1 shows the purification of mature rVWF on a cation exchanger presented in Example 1.

In fig. 2 shows a table of cleaning results.

In fig. 3 shows a silver-stained protein gel and Western blot illustrating the separation of mat-VWF and r-VWF propeptide (rVWF-PP) by the method of Example 1.

In fig. Figure 4 shows a block diagram of the experimental setup for Examples 2 and 3.

In fig. Figure 5 shows the chromatogram for example 2 and the chromatography scheme used for examples 2 and 3.

In fig. Figure 6 shows a table of the reagents used and a table of results for example 2.

In fig. Figure 7 shows another chromatogram from example 2 and a table of results for example 3.

In fig. 8 shows a silver-stained protein gel illustrating the separation of mat-rVWF and rVWF propeptide (rVWF-PP) by the method of Example 2 and Example 3.

In fig. 9 is a Western blot illustrating the separation of rVWF and rVWF propeptide by the method of Example 2 and Example 3.

In fig. 10 shows the chromatogram, chromatography scheme and buffer compositions for Example 4.

In fig. Figure 11 shows a table of results for example 4.

In fig. 12 shows a silver-stained protein gel and Western blot illustrating the separation of mat-rVWF and rVWF propeptide (rVWF-PP) by the method of Example 4.

In fig. 13 shows the chromatogram, chromatography scheme and buffer compositions for Example 5.

In fig. Figure 14 shows a table of results for example 5.

In fig. 15 shows the chromatogram, chromatography scheme and buffer compositions for Example 6.

In fig. Figure 16 shows a table of results for example 6.

In fig. 17 shows the chromatogram, chromatography scheme and buffer compositions for Example 7.

In fig. Figure 18 shows a table of results for example 7.

In fig. 19 shows the chromatogram, chromatography scheme and buffer compositions for Example 8.

In fig. Figure 20 shows a table of results for example 8.

In fig. 21 shows a silver-stained protein gel illustrating the separation of mat-rVWF and rVWF propeptide (rVWF-PP) by the method of Example 8.

In fig. 22 is a Western blot illustrating the separation of rVWF and rVWF propeptide by the method of Example 8. The 1% agarose gel demonstrates the multimeric structure of the products.

In fig. 23 shows a Western blot illustrating the separation of mat-rVWF and rVWF propeptide (rVWF-PP) by the method of Example 8.

In fig. Figure 24 shows the chromatogram, chromatography scheme, and buffer compositions for Example 9.

In fig. Figure 25 shows a table of results for example 9.

In fig. Figure 26 shows the product table for Example 9.

In fig. 27 is a silver-stained protein gel illustrating the separation of rVWF and rVWF propeptide by the method of Example 9.

In fig. 28 is a Western blot illustrating the separation of rVWF and rVWF propeptide by the method of Example 9. The 1% agarose gel demonstrates the multimeric structure of the products.

In fig. 29 is a Western blot illustrating the separation of rVWF and rVWF propeptide by the method of Example 9.

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In fig. 30 shows the purity of the product containing fractions obtained from the enhanced cation exchange chromatography (CEX) used in Examples 1, 2, 3, 6, 8 and 9.

In fig. 31 shows the removal rate of product-related impurities for Examples 1, 2, 3, 6, and 9.

In fig. 32 shows the purity of the product containing the enhanced size exclusion chromatography (SEC) fractions used in Examples 4 and 5.

In fig. 33 shows the removal rate of product-related impurities for Examples 4 and 5.

In fig. 34 shows the buffer compositions and materials used in the TMAE separation method.

In fig. 35 shows loading conditions for the mature VWF/VWF-propeptide complex treated with furin.

In fig. 36 shows details of the buffers, conditions, parameters and flow rates of the chromatographic method.

In fig. 37 shows a chromatogram of the dissociation of furin-treated mature VWF/VWF-propeptide complex into mature VWF and VWF-propeptide (VWF-PP). It shows the removal of VWF-PP from the fraction containing mature VWF.

In fig. 38 shows another separation chromatogram of mature VWF and VWF-propeptide (VWF-PP). It shows the removal of VWF-PP from the fraction containing mature VWF.

In fig. 39A and 39B are schematic diagrams of typical processes for purifying mature VWF, including separation of mature VWF and VWF-PP.

In fig. 40 is a table highlighting some of the advantages of the cation exchange chromatography method described herein.

In fig. 41 is a diagram of two chromatograms showing the separation of rVWF propeptide using size exclusion chromatography described herein using either SQA Running Buffer or SQC Running Buffer, which contains citrate. Changing the SEC parameters (SEC buffers) did not change the purification of mature VWF, except for increasing the removal/separation of residual VWF-PP.

In fig. 42 is a table showing some of the benefits of the optimized SEC buffer (SQC buffer). SQC buffer contains at least one chelating agent and has been shown to reduce the amount of VWF-PP in the purified mature VWF fraction.

In fig. 43A and 43B are block diagrams of post-processing protocols for rVWF. In fig. 43A shows the process currently in use. In fig. 43B shows the process described herein, which includes an improved CAT stage (UNO_S).

In fig. 44 shows a table of CAT stage chromatography equipment in the first generation (Gen 1) and second generation (Gen 2) process.

In fig. Figure 45 shows a table of washing and elution conditions for the Gen 2 process.

In fig. Figure 46 shows a comparison table of small-scale post-treatment stages of 1st and 2nd generation rVWF on UNO_Sphere S (CAT stage).

In fig. 47 shows a table of the cleaning and disinfection procedure for the UNO_Sphere S column.

In fig. Figure 48 shows a table of the composition of buffers for the CAT purification stage.

In fig. 49A and 49B show chromatograms of run VW_USS_05. In fig. 49A shows the entire chromatogram including the CIP procedure. In fig. 49B shows a 36% buffer B wash and gradient elution phase. UV absorption is shown in blue (280 nm) and magenta (254 nm).

In fig. Figure 50 shows an SDS-PAGE silver stain gel and Western blot of run VW_UUS_05.

In fig. 51 shows a multimer agarose gel from run VW_UUS_05.

In fig. 52 shows rVWF:Ag data from various study runs.

In fig. 53 shows Risto Co rVWF activity data from various assay runs.

In fig. 54 shows propeptide concentration data (propeptide (μg/mg rVWF:Ag)) for various assay runs.

In fig. 55 shows propeptide concentration data (propeptide (PP μg/1000 Risto U)) for various assay runs.

In fig. Figure 56 shows the key analytical results for the eluate pools of the various assay runs.

In fig. Figure 57 shows the CAT-E target criteria for successful method development.

In fig. 58 shows exemplary embodiments of anion exchange, cation exchange and size exclusion chromatography methods for us to separate mat-rVWF and rVWF-PP.

In fig. 59 shows the various forms of VWF: pro-VWF (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 called rVWF-PP).

In fig. 60 shows the nucleic acid and amino acid sequences of VWF.

In fig. 61 shows a Western blot of DF3338/042 and input data for analysis.

In fig. Figure 62 shows a Western blot of DF3362/023 and input data for analysis.

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In fig. 63 shows a comparison of the data with FIG. 61 and fig. 62.

In fig. 64 shows the amino acid sequence of a typical VWF-FVIII fusion protein, where the active FVIII is inserted into the VWF motif (VWF 764-1336 - FVIII heavy chain 24-760 - VWF 2218-2593 - FVIII light chain 1333-2351 - VWF 2620 to 2813).

In fig. 65 shows the amino acid sequence of a typical VWF-FVIII fusion protein, where the n-glycosylation-rich domain replaces the FVIII-B domain (FVIII heavy chain 19-760 - vWF 22182593 - FVIII light chain 1333-2351).

In fig. 66 is a table of buffers and compositions used in the vWF purification process embodiment described in Example 14.

In fig. Figure 67 shows a chromatogram and a diagram of the chromatogram of the VW_USS_07 cycle.

In fig. Figure 68 shows the main analytical results of the run.

In fig. 69 shows the silver staining SDS-PAGE gel of a representative run. Removal of rvWF propeptide was observed during wash steps Wash 1, WSD and Wash 2.

In fig. 70 is a table of buffers and compositions used in the embodiment of the vWF purification process described in Example 15. This example provides an alternative embodiment for separating the r-vWF propeptide from the r-VWF polypeptide after furin cleavage to test for additional sialylation.

In fig. 71 shows a chromatogram and a diagram of the chromatogram of the VW_USS_06 cycle.

In fig. Figure 72 shows the main analytical results of the run.

In fig. 73 shows the silver staining SDS-PAGE gel of a representative run.

In fig. 74 is a table of buffers and compositions used in the embodiment of the vWF purification process described in Example 16.

In fig. Figure 75 shows a chromatogram and a diagram of the chromatogram of the VW_USS_08 cycle.

In fig. 76 shows the main analytical results of the run including sialylation of the product.

In fig. 77 shows the DFM07247 silver stain gel in SDS-PAGE of a representative run.

In fig. 78 shows the sialylation profiles of eluates from runs VW_USS_06 and VW_USS_08.

Detailed description of the invention

Introduction.

The method described herein separates mature VWF and VWF propeptide that have been dissociated from a non-covalently bound heterodimeric complex containing mature VWF and VWF propeptide. This separation is facilitated (induced) by adding at least one chelating agent and/or increasing the pH to at least 7.0 of a solution containing mature VWF and VWF propeptide in a protein separation process. All of the enhanced anion exchange (AEX), cation exchange (CEX) and/or size exclusion chromatography (SEC) methods described herein can be combined in any manner to produce r-vWF with improved properties.

Selected definitions.

The term recombinant, when used with reference to, for example, a cell or 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 by alteration of the native nucleic acid or protein, or that the cell is derived from a cell modified in this way. Thus, for example, recombinant cells express genes that are not found in the native (non-recombinant) form of the cell, or express native genes that are otherwise abnormally expressed, underexpressed, or not expressed at all.

As used herein, the terms recombinant VWF or rVWF include VWF produced using recombinant DNA technology. In certain embodiments, the rVWF proteins of this invention may comprise a construct such as that described in US Pat. No. 8,597,910, which is incorporated herein by reference with respect to methods for producing recombinant VWF. VWF in this invention may include all potential forms, including monomeric and multimeric forms. It should also be understood that the present invention covers various forms of VWF that can be used in combination. For example, the VWF of this invention may include various multimers, various derivatives, as well as both biologically active derivatives and derivatives that are not biologically active.

In the context of this invention, recombinant VWF includes any member of the VWF family from, for example, a mammal, such as a primate, human, monkey, rabbit, pig, rodent, mouse, rat, hamster, gerbil, dog, cat, and biologically active derivatives thereof. Also covered are mutant and variant VWF proteins having activity, as well as functional fragments and fusion proteins based on VWF proteins. In addition, the VWF of the invention may further contain tags that facilitate cleaning, detection, or both. The VWF described herein may be further modified with a therapeutic moiety or a moiety suitable for in vitro or in vivo imaging.

The term VWF multimer refers to VWF containing at least 10 subunits or

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12, 14 or 16 subunits to about 20, 22, 24 or 26 subunits or more. The term subunit refers to the VWF monomer. As is known in the art, typically VWF dimers polymerize to form higher order multimers (see, e.g., Turecek et al., Semin. Thromb. Hemost, 2010, 36(5): 510-521 which is incorporated herein in its entirety description by reference for all purposes and in particular for all ideas regarding the analysis of VWF multimers).

The term VWF prepropeptide, prepro-VWF, or pro-VWF refers to an immature VWF polypeptide containing a signal peptide of about 22 amino acid residues, a VWF propeptide of about 741 amino acid residues, and a mature VWF subunit of about 2050 amino acid residues. Pro-VWF subunits can dimerize through disulfide bonds near their carboxyl termini in the endoplasmic reticulum to form tail-to-tail dimers, which are then transported to the Golgi apparatus. In the Golgi apparatus, additional head-to-head disulfide bonds are formed near the amino termini of the subunits, thereby forming multimers. Proteolytic cleavage of the VWF propeptide occurs through processing by the protease furin, resulting in the formation of the mature VWF/VWF-PP complex. When the letter r is included before the designation VWF, it indicates the recombinant version. In some embodiments, the methods described herein are applicable to recombinant VWF (rVWF).

The term VWF complex or mat-VWF/VWF-PP complex refers to a non-covalently associated heterodimeric structure containing the mature VWF subunit and the VWF propeptide. The VWF complex can be formed as a furin cleavage product between the propeptide portion and the mature VWF portion of the VWF pre-propeptide. When the letter r is included before the designation VWF, it indicates the recombinant version. In some embodiments, the methods described herein are applicable to recombinant VWF (rVWF).

The term mature VWF or mat-VWF refers to the mature VWF subunit, consisting of about 2050 amino acid residues. The mature VWF subunit may be part of the VWF pre-propeptide or the VWF complex. Mature VWF can be called free VWF after separation (isolation) from the VWF propeptide. When the letter r is included before the designation VWF, it indicates the recombinant version. In some embodiments, the methods described herein are applicable to recombinant VWF (rVWF).

The term VWF propeptide or VWF-PP refers to the VWF propeptide consisting of approximately 741 amino acid residues. The VWF propeptide may be part of a VWF pre-propeptide or a VWF complex. For example, in the VWF complex, the VWF propeptide is noncovalently associated with the mature VWF subunit. VWF propeptide may be called free VWF propeptide after separation (isolation) from mature VWF. When the letter r is included before the designation VWF, it indicates the recombinant version. In some embodiments, the methods described herein are applicable to recombinant VWF (rVWF).

The terms isolated, purified or biologically pure refer to a material that is substantially or substantially free of the components that normally accompany it in its native state. Purity and uniformity are usually determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. VWF is the predominant species present in the preparation, which is essentially purified. The term purified, in some embodiments, means that the nucleic acid or protein produces substantially a single band on an electrophoretic gel. In other embodiments, this means that the nucleic acid or protein is at least 50% pure, more preferably at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% pure, or more. To clean or purify in other embodiments means to remove at least one contaminant from the composition being purified. In this sense, purification does not require that the purified compound be homogeneous, for example 100% pure.

As used herein, the term "about" means an approximate range of plus or minus 10% of the stated value. For example, a formulation of about 20% covers the range of 18-22%.

Detailed Description of Embodiments

This invention relates to a method for producing a highly purified composition containing free mature recombinant von Willebrand factor (rVWF), comprising the steps of: dissociating the mature rVWF from the rVWF propeptide using a solution (for example, a dissociation solution) containing at least one chelating agent or having a pH at least 7; separating the free mature rVWF from the rVWF propeptide, and collecting a free mature rVWF composition containing at least 95% free mature rVWF and less than 5% rVWF propeptide.

The method of this invention is particularly suitable for separating mature VWF from VWF propeptide in vitro. In some embodiments, separation is induced by adding one or more chelating agents to a solution containing mature VWF and VWF-PP, increasing the pH of the solution to at least 7.0, or a combination thereof. In some embodiments, the pH is increased to a range of pH 7.0 to pH 9.0.

The separation method may involve the use of one or more protein separation techniques, such as, but not limited to, chromatographic techniques for isolating mature VWF from VWF-PP. Using this method, a free, mature rVWF composition of high purity can be obtained. 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 complex. In some cases, the free mature rVWF composition contains at least 96% free mature rVWF and less than 4% free rVWF-PP and/or matVWF/VWF-PP complex, at least 97% free mature rVWF and less than 3% free rVWF-PP and/or matVWF/VWF-PP complex, at least 98% free mature rVWF and less than 2% free rVWF-PP and/or matVWF/VWFPP complex, at least 99% free mature rVWF and less than 1% free rVWF-PP and/or matVWF/VWF-PP complex, at least 99.5% free mature rVWF and less than 0.5% free rVWF-PP and/or matVWF/VWF-PP complex.

Purification using anion exchange chromatography

In one aspect of this method, mature rVWF (mat-rVWF) is separated from rVWF-PP using anion exchange chromatography (AEX). In some cases, remaining host cell-derived contaminants such as CHO host cell proteins, process contaminants such as recombinant furin and small molecule virus inactivation reagents, media compounds such as soy peptone, and other impurities associated with products are removed from the mature VWF.

In another aspect of this method, mature rVWF-PP is separated from rVWF-PP, such as residual rVWF-PP or free rVWF-PP, using anion exchange chromatography. For separation, the feed composition, feed solution, or feed composition may have a low pH and at least one chelating agent. The loading composition can be applied to an anion exchanger operating in flow 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 coupling 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 greater, for example, pH 7.0 to pH 9.0. For example, the gradient elution buffer may contain EDTA and have a pH of 8.5.

In some embodiments, the present invention provides a method for preparing a composition containing highly purified mature recombinant propeptide-depleted rVWF (high purity matrVWF), the method comprising the steps of: (a) loading a solution containing pro-rVWF, mat-rVWF/rVWF complex -PP, mat-rVWF and/or rVWF propeptide (rVWF-PP), onto an anion exchange column, wherein said pro-rVWF, mat-rVWF/rVWF-PP complex and mat-rVWF are bound to said anion exchange column; (b) washing said anion exchange column in step a) containing said bound pro-rVWF, mat-rVWF/rVWF-PP complex and mat-rVWF, with one or more wash buffers; (c) treating said column in b) containing bound pro-rVWF, the mat-rVWF/rVWF-PP and mat-rVWF complex, with furin, wherein said furin cleaves said pro-rVWF into mat-rVWF and rVWF-PP; (d) eluting said bound pro-rVWF, the matrVWF/rVWF-PP complex and mat-rVWF from the column in c) with an elution buffer, wherein said elution buffer induces dissociation of said rVWF-PP from mat-rVWF non-covalently bound to said rVWF-PP wherein said dissociation is induced by: (i) adding at least one chelating agent to said elution buffer, or (ii) increasing the pH of said elution buffer to a pH of at least 7; and (e) collecting said mat-rVWF separately from said rVWF-PP to produce a highly purified mat-rVWF composition, wherein said highly purified mat-rVWF composition contains at least 95% mature rVWF and less than 5% rVWF-PP.

In some embodiments, a) and b) are performed simultaneously in the same step. In some embodiments, the solution in a) contains a stream from an immunoaffinity purification method. In some embodiments, the solution in a) includes a flow-through solution from a monoclonal antibody column, wherein said monoclonal antibody is a FVIII monoclonal antibody. In some embodiments, the solution in a) is selected from the group consisting of a cell culture medium, an antibody column flow-through solution, and a buffered solution.

In some embodiments, step (b) washing said anion exchange column in a) containing said bound pro-rVWF, mat-rVWF/rVWF-PP complex and mat-rVWF involves washing with one or more wash buffers, wherein one or more washes with buffer includes one, two, three, four and/or five wash buffers. In some embodiments, the second wash buffer contains components for viral inactivation. In some embodiments, when four or five wash buffers are used, the second wash buffer contains components for viral inactivation. In some embodiments, when four or five wash buffers are used, the second or third wash buffer contains

- 9 045553 components for viral inactivation. 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 buffers 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 7 to 8, and the fourth wash buffer has a pH of about 5 to 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 treatment step is performed with a buffer that has a pH greater than the fourth wash buffer. In some embodiments, when four wash buffers are used, a virus inactivation processing step is applied 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 treatment step is performed with a buffer that has a pH greater than the third wash buffer. In some embodiments, the virus inactivation step occurs with a buffer that has 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 7.4 to 7.5, and the third wash buffer has a pH of about 5.5.

Anion exchange chromatography can be performed as is known to 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™, EmphaseTM 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-action AEX resins (e.g. Capto Adhere, Capto adhere impress, or MEP Hypercell), as well as any DEAE, TMAE, tertiary or quaternary amine or PEL resins In some embodiments implementation of the anion exchanger is a membrane anion exchanger. In some embodiments, the membrane anion exchanger includes, without limitation, Sartobind Q®, Sartobind STIC® PA, Mustang Q®, or ChromaSorb®. In some embodiments, the anion exchanger is a Fractogel TMAE column (Merck-Millipore) or equivalent.

In some embodiments, the loading concentration of pro-VWF is from about 90 IU/ml to about 270 IU/ml resin, for example, 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 270 IU/ml, from about 140 IU/ml to about 270 IU/ml, from about 150 IU/ml to about 270 IU/ml, from about 90 IU/ml to about 250 IU/ml, from about 100 IU/ml to about 250 IU/ml, from about 110 IU/ml to about 250 IU/ml, from about 120 IU/ml to about 250 IU/ml, from about 130 IU/ml to about 250 IU/ml, from about 130 IU/ml ml to about 250 IU/ml, from about 140 IU/ml to about 250 IU/ml, from about 150 IU/ml to about 250 IU/ml, from about 90 IU/ml to about 200 IU/ml, from about 100 IU/ml to about 200 IU/ml, from about 110 IU/ml to about 200 IU/ml, from about 120 IU/ml to about 200 IU/ml, from about 130 IU/ml to about 200 IU/ml, from about 130 IU/ml to about 200 IU/ml, from about 140 IU/ml to about 200 IU/ml, from about 150 IU/ml to about 200 IU/ml, from about 90 IU/ml to about 100 IU/ml , from about 100 IU/ml to about 150 IU/ml, from about 150 IU/ml to about 200 IU/ml, from about 200 IU/ml to about 250 IU/ml or from about 250 IU/ml to about 270 IU /ml resin.

In some embodiments, the anion exchange method includes a buffer system. In some embodiments, the buffer system contains one or more elution buffers. In some embodiments, the buffer system contains 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 contains at least two elution buffers and at least two wash buffers.

In some embodiments, the concentration of the loading solution is from about 90 to about 270 IU/mL of resin, for example, from about 90 to about 270 IU/mL, from about 100 to about 270 IU/mL, from about 110 to about 270 IU/mL , from about 120 to about 270 IU/ml, from about 130 to about 270

IU/ml, about 130 to about 270 IU/ml, about 140 to about 270 IU/ml, about 150 to about 270

IU/ml, from about 90 to about 250 IU/ml, from about 100 to about 250 IU/ml, from about 110 to about 250

IU/ml, about 120 to about 250 IU/ml, about 130 to about 250 IU/ml, about 130 to about 250

IU/ml, about 140 to about 250 IU/ml, about 150 to about 250 IU/ml, about 90 to about 200

- 10 045553

IU/ml, from about 100 to about 200 IU/ml, from about 110 to about 200 IU/ml, from about 120 to about 200

IU/ml, from about 130 to about 200 IU/ml, from about 130 to about 200 IU/ml, from about 140 to about 200

IU/ml, about 150 to about 200 IU/ml, about 90 to about 100 IU/ml, about 100 to about 150

IU/ml, about 150 to about 200 IU/ml, about 200 to about 250 IU/ml, or about 250 to about

270 IU/ml resin.

In some embodiments, the pH of the feed composition, feed solution, or feed composition 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, for example, pH 6.0-pH 9.0, pH 6.3-pH 9.0, pH 6.5-pH 9.0, pH 7.0-pH 9.0, pH 7.5 -pH 9.0, pH 7.7, pH 9.0, pH 8.0, pH 9.0, pH 6.0, pH 8.5, pH 6.5, pH 8.5, pH 7 ,0-pH 8.5, pH 7.5-pH 8.5, pH 6.0-pH 8.0, pH 6.5-pH 8.0, pH 7.0-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 or pH 9.0.

In some embodiments, the conductivity of the feed composition, feed solution, or feed composition comprising pro-rVWF, mat-rVWF/rVWF-PP complex, mat-rVWF, and/or rVWF propeptide (rVWF-PP) is from about 5 to about 40 mS /cm, for example, from about 5 to about 40 mS/cm, from about 10 to about 40 mS/cm, from about 15 to about 40 mS/cm, from about 20 to about 40 mS/cm, from about 25 to about 40 mS/cm, about 30 to about 40 mS/cm, about 10 to about 40 mS/cm, about 10 to about 30 mS/cm, about 5 to about 15 mS/cm, about 15 to about 30 mS/cm or from about 20 to about 40 mS/cm.

In some embodiments, 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 diluted with a buffer containing sodium citrate, such as , without limitation, 10-80 mM sodium citrate, 15-80 mM sodium citrate, 10-80 mM sodium citrate, 15-60 mM sodium citrate, 20-60 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, or the like.

In some embodiments, the first wash buffer contains 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 has a pH in the range of pH 6.0 to pH 9.0 and optionally contains at least one chelating agent. In some embodiments, the first wash buffer has a pH in the range of pH 6.0 to pH 6.9. In some embodiments, the second wash buffer has a pH in the range of pH 7.0 to pH 9.0. In some embodiments, the first wash buffer may contain 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 of less than 7. In one embodiment, the second wash buffer has a pH of greater than 7. In some embodiments, when two wash buffers are used, the first wash buffer has a pH of less than 7, and the second wash buffer has a pH of less than 7. more than 7.

In some embodiments, one or more wash buffers contain NaCl at a concentration of 120-200 mM, 130-200 mM, 140-200 mM, 150-200 mM, 120-190 mM, 130-190 mM, 140-190 mM, 150- 190 mM, 120-180 mM, 130-180 mM, 140-180 mM, 150-180 mM, 120 mM, 125 mM, 130 mM, 135 mM, 140 mM, 145 mM, 150 mM, 155 mM, 1 60 mM, 165 mM, 170 mM, 175 mM, 180 mM, 185 mM, 190 mM, 195 mM or 200 mM.

In some embodiments, the starting composition, feed solution, or feed composition containing mature VWF and VWF-PP is contacted with a buffer containing at least one chelating agent, and optionally a buffer having a pH in the range of pH 6.0 to pH 9 ,0. In some embodiments, the feed composition, feed solution, or feed composition is contacted with a buffer having a pH in the range of pH 6.0 to pH 9.0, and optionally a buffer containing 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 wash buffer. In some embodiments, the buffer is an elution buffer. In some embodiments, the buffer is a wash buffer with a pH of 6.0 to 6.9. In some embodiments, the buffer is an elution buffer with a pH of 7.0 to 9.0. In some embodiments, the starting composition, loading solution, or loading composition containing mature VWF and VWF-PP is contacted first with a wash buffer having a pH of 6.0 to 6.9, and then with at least one elution buffer, having a pH from 7.0 to 9.0.

In some embodiments, the mature VWF is eluted in an anion exchange chromatography step using a single elution buffer. In some embodiments, the mature VWF is eluted in an anion exchange chromatography step using a gradient elution method including more than one elution buffer. For example, elution can be performed using two elution buffers, such as, for example, a first elution buffer and a second elution buffer. In some embodiments, the first elution buffer

- 11 045553 contains 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 contains 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 may contain 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 of less than 7. In one embodiment, the second elution buffer has a pH of greater than 7. In some embodiments, when two elution buffers are used, the first elution buffer has a pH of less than 7 and the second elution buffer has a pH of more than 7.

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-pH 9.0, pH 6.3-pH 9.0, pH 6.5- pH 9.0, pH 7.0 pH 9.0, pH 7.5-pH 9.0, pH 7.7.0-pH 9.0, pH 8.0-pH 9.0, pH 6.0- pH 8.5, pH 6.5-pH 8.5, pH 7.0-pH 8.5, pH 7.5-pH 8.5, pH 6.0-pH 8.0, pH 6.5-pH 8 ,0, pH 7.0-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. In some embodiments, it contains two elution buffers, for example, a first and a second elution buffer.

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-pH 9.0, pH 6.3-pH 9.0, pH 6.5- pH 9.0, pH 7.0 pH 9.0, pH 7.5-pH 9.0, pH 7.7.0-pH 9.0, pH 8.0-pH 9.0, pH 6.0- pH 8.5, pH 6.5-pH 8.5, pH 7.0-pH 8.5, pH 7.5-pH 8.5, pH 6.0-pH 8.0, pH 6.5-pH 8 ,0, pH 7.0-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. In some embodiments, it contains two elution buffers, for example, a first and a second elution buffer.

In some embodiments, the pH of the elution buffer is increased compared to the original solution in step a), increased compared to the first elution buffer when two elution buffers are used, and/or increased compared to the wash buffer when 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 the 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 has a pH greater than 7. In some embodiments, when two wash buffers and two elution buffers are used, the wash buffers and the first elution buffer have a pH less than 7 and the second elution buffer the buffer has 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, the first wash buffer has a pH less than 7, and the second wash buffer, and both elution buffers have a pH greater than 7.

In some embodiments, the pH of one or more wash 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 a loading solution containing pro-rVWF, mat-rVWF/rVWF-PP complex, mat-rVWF and/or rVWF propeptide (rVWF-PP) as specified 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 cause the dissociation of the mat-rVWF/rVWF-PP complex into solution in step (a) of the process to mat-rVWF and rVWF-PP, said dissociation occurring due to the destruction of the non-covalently bound mat-rVWF and rVWFPP. In some embodiments, the pH of the feed solution is increased from at least about 7.2 to about 7.8. In some loading solution embodiments, the pH of the loading solution is increased to at least about 7.6. In some embodiments, the pH of the feed solution is increased by adding basic amino acids. 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 from 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 adding basic amino acids. In some embodiments, the one or more wash buffers have a pH of at least 7. In some embodiments, the pH of the one or more elution buffers is increased from 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 adding basic amino acids. In some embodiments, the one or more elution buffers have a pH of at least 7.

In some embodiments, one or more buffers (including wash and/or elution buffers) contain one or more chelating agents. In some embodiments, the elution buffer contains at least one chelating agent. The chelating agent may 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 NTC, DTPA, EDDS, EDTA, EGTA, CDTA, and citrate. In some embodiments, the chelating agent is HTC. In some embodiments, the chelating agent is DTPC. 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 at b) contain said one or more chelating agents, and have a pH of at least 7.

In some embodiments, one or more buffers (including wash and/or elution buffers) contain sodium citrate in a range including, but not limited to, 10 mM-80 mM sodium citrate, 15 mM-80 mM sodium citrate, 10 mM-80 mM citrate sodium, 15 mM-60 mM 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, or the like.

In some embodiments, the first elution buffer further contains sodium citrate in a range including, but not limited to, 10-60 mM sodium citrate, 15-60 mM sodium citrate, 10-50 mM sodium citrate, 15-50 mM sodium citrate, 20-60 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, or the like.

In some embodiments, the second elution buffer further contains sodium citrate, such as, but not limited to, 10-60 mM sodium citrate, 15-60 mM sodium citrate, 10-50 mM sodium citrate, 15-50 mM 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, 60 mM sodium citrate, or the like.

In some embodiments, elution buffer A and/or elution buffer B in the anion exchange chromatography step contains from about 0.5 to about 20 mM EDTA, for example, from about 0.5 to about 20 mM, from about 1 to about 20 mM, from about 1.5 to about 20 mM, about 2 to about 20 mM, about 3 to about 20 mM, about 5 to about 20 mM, about 0.5 to about 15 mM, about 1 to about 10 mM , about 1 mM to about 5 mM, about 5 mM, about 0.5 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, or the like

In some embodiments, citrate may be detected in the eluent after removal of the rVWF propeptide using an anion exchange elution method. In some embodiments, citrate may be detected in the eluent after removal of the rVWF propeptide using a stepwise anion exchange elution method. In some embodiments, citrate may be detected in the eluent after removal of the rVWF propeptide using an anion exchange gradient elution method. In some embodiments, the anion exchange counterion is citrate ^3- .

Any of the buffers (buffer systems) described herein can be selected from the group consisting of glycine, HEPES (4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid), Tris-HCl (Tris(hydroxymethyl)aminomethane), histidine, imidazole, citrate acetate, citrate, acetate, solvate, phosphate, tris-HCl, bis-tris, histidine, imidazole, arginine HCl, lysine HCl and 2-(Nmorpholino)ethanesulfonic acid as separate buffers or as a combination of two or more buffers. In some embodiments, the buffer contains glycine. In some embodiments, the buffer contains HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). In some embodiments, the buffer contains Tris-HCl (Tris(hydroxymethyl)-aminomethane). In some embodiments, the buffer contains histidine. In some embodiments, the buffer

- 13 045553 contains imidazole. In some embodiments, the buffer contains acetate citrate. In some embodiments, the buffer contains citrate. In some embodiments, the buffer contains acetate. In some embodiments, the buffer contains MES. In some embodiments, the buffer contains phosphate. In some embodiments, the buffer contains Tris-HCl. In some embodiments, the buffer contains bis-Tris. In some embodiments, the buffer contains histidine. In some embodiments, the buffer contains an imidazole. In some embodiments, the buffer contains arginine HCl. In some embodiments, the buffer contains lysine HCl. In some embodiments, the buffer contains 2-(Nmorpholino)ethanesulfonic acid. In some embodiments, the buffer comprises one, two, three, or four of the buffers described herein.

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

In some embodiments, the one or more buffers comprise at least one buffer having a conductivity of >0.5 mS/cm at 25°C. In some embodiments, the one or more buffers comprise at least one buffer having 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 having 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 having 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 having 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 having 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 having 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 having a conductivity of 2.0 ± 0.2 mS/cm at 25°C.

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

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

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

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 contain one or more non-reducing sugars. In some embodiments, the non-reducing sugar includes, but is not limited to, sucrose, trehalose, mannitol, sorbitol, galactitol, and/or xylitol. In some embodiments, the one or more buffers further contain one or more sugar alcohols. In some embodiments, the one or more buffers further comprise one or more polyols. In some embodiments, the sugar alcohol or polyol includes, without limitation, mannitol, xylitol, erythritol, threitol, sorbitol and/or glycerol. In some embodiments, the buffers further comprise ethylene glycol, propylene glycol, glycerol, 1,2,3-propanetriol), meso-erythritol, and/or erythritol (meso-1,2,3,4-butantetrol).

In some embodiments, the buffer may contain one or more monovalent cations. In some embodiments, the one or more monovalent cations are selected from the group consisting of Na ⁺ , K ⁺ , Li ⁺ , Cs ⁺ and NH ₄ ⁺ . For example, a monovalent cation may represent

- 14 045553 is Na ⁺ . In other embodiments, the buffer contains one or more monovalent, divalent and/or trivalent anions. One or more monovalent, divalent and/or trivalent anions may be selected from the group consisting of Cl ^- , acetate - , SO4 ^{2 -} , Br ^- , citrate ^{3 -} , PO4 ^{3 -} and BO ₃ ^{3 -} . In some embodiments, the buffer contains 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 contain 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 contain one or more monovalent, divalent and/or trivalent anions. In some embodiments, one or more monovalent, divalent and/or trivalent anions is selected from the group consisting of Cl ^- , acetate ^- , SO4 ² - , Br ^- and citrate ^{3 -} .

The pH of any of the buffers can be adjusted (increased) by adding amino acid, Tris, NaOH, ethanolamine, etc.

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

Purification by cation exchange chromatography

In one aspect of this method, mature VWF (matVWF) is separated from VWF-PP using cation exchange chromatography (CEX). In some cases, remaining host cell-derived contaminants such as CHO host cell proteins, process contaminants such as recombinant furin and small molecule virus inactivation reagents, media compounds such as soy peptone, and other impurities associated with products are removed from the mature VWF.

In another aspect of this method, mature VWF is separated from VWF-PP, such as residual VWFPP or free VWF-PP, using cation exchange chromatography. For separation, the feed composition, feed solution, or feed composition may have a low pH and at least one chelating agent. In some embodiments, the starting composition, loading solution, or loading composition comprises pro-rVWF, mat-rVWF/rVWF-PP complex, mat-rVWF, and/or rVWF propeptide (rVWF-PP). In some embodiments, the cation exchanger is operated in coupling 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 greater, for example, pH 7.0 to pH 9.0. For example, the gradient elution buffer may contain EDTA and have a pH of 8.5.

In some embodiments, the present invention provides a method for preparing a composition containing highly purified mature recombinant propeptide-depleted rVWF (high purity matrVWF), the method comprising the steps of: (a) loading a solution containing pro-rVWF, mat-rVWF/rVWF complex -PP, mat-rVWF and/or rVWF propeptide (rVWF-PP), onto a cation exchange column, wherein said pro-rVWF, mat-rVWF/rVWF-PP complex and mat-rVWF are bound to said cation exchange column; (b) washing said cation exchange column in step a) containing said bound pro-rVWF, mat-rVWF/rVWF-PP complex and mat-rVWF, with one or more wash buffers; (c) treating said column in b) containing bound pro-rVWF, the mat-rVWF/rVWF-PP and mat-rVWF complex, with furin, wherein said furin cleaves said pro-rVWF into mat-rVWF and rVWF-PP; (d) eluting said bound pro-rVWF, the matrVWF/rVWF-PP complex and mat-rVWF from the column in c) with an elution buffer, wherein said elution buffer induces dissociation of said rVWF-PP from mat-rVWF non-covalently bound to said rVWF-PP wherein said dissociation is induced by: (i) adding at least one chelating agent to said elution buffer, or (ii) increasing the pH of said elution buffer to a pH of at least 7; and (e) collecting said mat-rVWF separately from said rVWF-PP to produce a highly purified mat-rVWF composition, wherein said highly purified mat-rVWF composition contains at least 95% mature rVWF and less than 5% rVWF-PP.

In some embodiments, a) and b) are performed simultaneously in the same step. In some embodiments, the solution in a) contains a stream from an immunoaffinity purification method. In some embodiments, the solution in a) comprises a flow-through solution from a monoclonal antibody column, wherein said monoclonal antibody is a FVIII monoclonal antibody. In some embodiments, the solution in a) is selected from the group consisting of cell culture medium, antibody column flow-through solution, and a buffered solution.

The cation exchanger can be operated in coupling mode to separate mature VWF and VWF-PP. Cation exchange chromatography can be performed as known to those skilled in the art. In some embodiments, the cation exchanger includes, but is not limited to, POROS®

- 15 045553

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), UNOsphereS (Bio-rad, Hercules, CA), MacroprepCM ((Bio-rad, Hercules, CA), Fractogel EMD SO3 (Merck), Fractogel EMD COO (Merck), Fractogel EMD SE Hicap (Merck), Cellufine Sulfate (JNC), CM and SP Trisacryl (Pall), CM and S HyperD (Pall), S and CM Sepharose CL (Ge Healthcare), S and CM Sepharose FF (GE Healthcare), S and CM CAPTO™ (GE Healthcare), MonoS (GE Healthcare), Source S (GE Healthcare), Nuvia S (Merck), or Cellufine phosphate (JNC). In some embodiments, the cation exchanger is a membrane cation exchanger. In some embodiments, the membrane cation exchanger includes, without limitation, Mustang S (Pall) or Sartobind® S. In some embodiments, the cation exchanger is a UNO_Sphere S column (Bio-Rad) or equivalent.

In some embodiments, step (b) washing said cation exchange column in a) containing said bound pro-rVWF, mat-rVWF/rVWF-PP complex and mat-rVWF involves washing with one or more wash buffers, wherein one or more washes with buffer includes one, two, three, four and/or five wash buffers. In some embodiments, the second wash buffer contains components for viral inactivation. In some embodiments, when four or five wash buffers are used, the second wash buffer contains components for viral inactivation. In some embodiments, when four or five wash buffers are used, the second or third wash buffer contains components for viral inactivation. 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 buffers 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 7 to 8, and the fourth wash buffer has a pH of about 5 to 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 treatment step is performed with a buffer that has a pH greater than the fourth wash buffer. In some embodiments, when four wash buffers are used, a virus inactivation processing step is applied 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 treatment step is performed with a buffer that has a pH greater than the third wash buffer. In some embodiments, the virus inactivation step occurs with a buffer that has 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 7.4 to 7.5, and the third wash buffer has a pH of about 5.5.

In some embodiments, the loading concentration of pro-VWF is from about 90 to about 270 IU/mL of resin, for example, from about 90 to about 270 IU/mL, from about 100 IU/mL to about 270 IU/mL, from about 110 to about 270 IU/ml, from about 120 to about 270 IU/ml, from about 130 to about 270 IU/ml, from about 130 IU/ml to about 270 IU/ml, from about 140 IU/ml to about 270 IU/ ml, from about 150 to about 270 IU/ml, from about 90 to about 250 IU/ml, from about 100 to about 250 IU/ml, from about 110 to about 250 IU/ml, from about 120 to about 250 IU/ ml, from about 130 to about 250 IU/ml, from about 130 to about 250 IU/ml, from about 140 to about 250 IU/ml, from about 150 to about 250 IU/ml, from about 90 to about 200 IU/ ml, from about 100 to about 200 IU/ml, from about 110 to about 200 IU/ml, from about 120 IU/ml to about 200 IU/ml, from about 130 IU/ml to about 200 IU/ml, from about 130 to about 200 IU/ml, from about 140 to about 200 IU/ml, from about 150 to about 200 IU/ml, from about 90 to about 100 IU/ml, from about 100 to about 150 IU/ml, from about 150 to about 200 IU/ml, about 200 to about 250 IU/ml, or about 250 to about 270 IU/ml resin.

In some embodiments, the cation exchange method includes a buffer system. In some embodiments, the buffer system contains one or more elution buffers. In some embodiments, the buffer system contains 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 contains at least two elution buffers and at least two wash buffers.

In some embodiments, the first wash buffer contains at least

- 16 045553 one chelating agent and optionally has a pH in the range from pH 6.0 to pH 9.0. In some embodiments, the first wash buffer has a pH in the range of pH 6.0 to pH 9.0 and optionally contains at least one chelating agent. In some embodiments, the first wash buffer has a pH in the range of pH 6.0 to pH 6.9. In some embodiments, the second wash buffer has a pH in the range of pH 7.0 to pH 9.0. In some embodiments, the first wash buffer may contain 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 buffer has a pH less than 7. In some embodiments, 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 .

In some embodiments, one or more wash buffers contain NaCl at a concentration of 120 mM - 200 mM, 130 mM - 200 mM, 140 mM - 200 mM, 150 mM - 200 mM, 120 mM - 190 mM, 130 mM - 190 mM, 140 mM - 190 mM, 150 mM - 190 mM, 120 mM - 180 mM, 130 mM - 180 mM, 140 mM 180 mM, 150 mM - 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.

In some embodiments, the starting composition, feed solution, or feed composition containing mature VWF and VWF-PP is contacted with a buffer containing at least one chelating agent, and optionally a buffer having a pH in the range of pH 6.0 to pH 9 ,0. In some embodiments, the feed composition, feed solution, or feed composition is contacted with a buffer having a pH in the range of pH 6.0 to pH 9.0, and optionally a buffer containing 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 wash buffer. In some embodiments, the buffer is an elution buffer. In some embodiments, the buffer is a wash buffer with a pH of 6.0 to 6.9. In some embodiments, the buffer is an elution buffer with a pH of 7.0 to 9.0. In some embodiments, a feed composition, feed solution, or feed composition comprising mature VWF and VWF-PP is contacted first with a wash buffer having a pH of 6.0 to 6.9 and then with at least one elution buffer, having a pH from 7.0 to 9.0.

In some embodiments, the mature VWF is eluted in an anion exchange chromatography step using a single elution buffer. In some embodiments, the mature VWF is eluted in an anion exchange chromatography step using a gradient elution method including more than one elution buffer. For example, elution can be performed using two elution buffers, such as, for example, a first elution buffer and a second elution buffer. In some embodiments, the first elution buffer contains 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 contains 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 may contain 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 some embodiments, 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 .

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-pH 9.0, pH 6.3-pH 9.0, pH 6.5- pH 9.0, pH 7.0-pH 9.0, pH 7.5-pH 9.0, pH 7.7.0-pH 9.0, pH 8.0-pH 9.0, pH 6, 0-pH 8.5, pH 6.5-pH 8.5, pH 7.0-pH 8.5, pH 7.5-pH 8.5, pH 6.0-pH 8.0, pH 6, 5-pH 8.0, pH 7.0-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. In some embodiments, it contains two elution buffers, for example, a first and a second elution buffer.

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-pH 9.0, pH 6.3-pH 9.0, pH 6.5- pH 9.0, pH 7.0 pH 9.0, pH 7.5-pH 9.0, pH 7.7.0-pH 9.0, pH 8.0-pH 9.0, pH 6.0- pH 8.5, pH 6.5-pH 8.5, pH 7.0-pH 8.5, pH 7.5-pH 8.5, pH 6.0-pH 8.0, pH 6.5-pH 8 ,0, pH 7.0-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. In some embodiments, it contains two elution buffers, for example, a first and a second elution buffer.

- 17 045553

In some embodiments, the pH of the elution buffer is increased compared to the original solution in step a), increased compared to the first elution buffer when two elution buffers are used, and/or increased compared to the wash buffer when 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 the 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 has a pH greater than 7. In some embodiments, when two wash buffers and two elution buffers are used, the wash buffers and the first elution buffer have a pH less than 7 and the second elution buffer the buffer has 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, the first wash buffer has a pH less than 7, and the second wash buffer, and both elution buffers have a pH greater than 7.

In some embodiments, the pH of one or more wash 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 a loading solution containing pro-rVWF, mat-rVWF/rVWF-PP complex, mat-rVWF and/or rVWF propeptide (rVWF-PP) as specified 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 cause the dissociation of the mat-rVWF/rVWF-PP complex into solution in step (a) of the process to mat-rVWF and rVWF-PP, said dissociation occurring due to the destruction of the non-covalently bound mat-rVWF and rVWFPP. In some embodiments, the pH of the feed solution is increased from at least about 7.2 to about 7.8. In some loading solution embodiments, the pH of the loading solution is increased to at least about 7.6. In some embodiments, the pH of the feed solution is increased by adding basic amino acids. 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 from 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 adding basic amino acids. In some embodiments, the one or more wash buffers have a pH of at least 7. In some embodiments, the pH of the one or more elution buffers is increased from 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 adding basic amino acids. In some embodiments, one or more elution buffers have a pH of at least 7.

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-pH 9.0, pH 6.3-pH 9.0, pH 6.5-pH 9.0, pH 7.0-pH 9.0, pH 7.5-pH 9.0, pH 7.7.0-pH 9.0, pH 8.0-pH 9.0, pH 6.0-pH 8.5, pH 6.5-pH 8.5, pH 7.0-pH 8, 5, pH 7.5-pH 8.5, pH 6.0-pH 8.0, pH 6.5-pH 8.0, pH 7.0-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 or pH 9.0.

In some embodiments, the conductivity of the feed composition, feed solution, or feed composition comprising pro-rVWF, mat-rVWF/rVWF-PP complex, mat-rVWF, and/or rVWF propeptide (rVWF-PP) is from about 5 to about 40 mS /cm, for example, from about 5 to about 40 mS/cm, from about 10 to about 40 mS/cm, from about 15 to about 40 mS/cm, from about 20 to about 40 mS/cm, from about 25 to about 40 mS/cm, about 30 to about 40 mS/cm, about 10 to about 40 mS/cm, about 10 to about 30 mS/cm, about 5 to about 15 mS/cm, about 15 to about 30 mS/cm or from about 20 to about 40 mS/cm.

In some embodiments, the loading solution containing pro-rVWF, matrVWF/rVWF-PP complex, mat-rVWF and/or rVWF propeptide (rVWF-PP) is diluted with a buffer containing sodium citrate, such as, but not limited to, 10 mM-80 mM sodium citrate, 15 mM-80 mM sodium citrate, 10 mM 80 mM sodium citrate, 15 mM-60 mM 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, or the like.

In some embodiments, the pH of the wash buffer for the cation exchange step

- 18 045553 chromatography is from pH 6.0 to pH 9.0, for example, pH 6.0-pH 9.0, pH 6.3-pH 9.0, pH 6.5-pH 9.0, pH

7.0-pH 9.0, pH 7.5-pH 9.0, pH 7.7,0-pH 9.0, pH 8.0-pH 9.0, pH 6.0-pH 8.5 , pH 6.5-pH 8.5, pH 7.0-pH 8.5, pH

7.5-pH 8.5, pH 6.0-pH 8.0, pH 6.5-pH 8.0, pH 7.0-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 or pH 9.0.

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-pH 9.0, pH 6.3-pH 9.0, pH 6.5- pH 9.0, pH 7.0 pH 9.0, pH 7.5-pH 9.0, pH 7.7.0-pH 9.0, pH 8.0-pH 9.0, pH 6.0- pH 8.5, pH 6.5-pH 8.5, pH 7.0-pH 8.5, pH 7.5-pH 8.5, pH 6.0-pH 8.0, pH 6.5-pH 8 ,0, pH 7.0-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 or pH 9.0.

In some embodiments, the pH of the elution buffer is increased compared to the original solution in step a), increased compared to the first elution buffer when two elution buffers are used, and/or increased compared to the wash buffer when 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, the first elution buffer has a pH less than 7 and the second elution buffer has a pH of less than 7. the 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 has a pH greater than 7. In some embodiments, when two wash buffers and two elution buffers are used, the two wash buffers and the first elution buffer have a pH less than 7 and the second elution buffer the buffer has 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, the first wash buffer has a pH less than 7, and the second wash buffer and both elution buffers have a pH greater than 7.

In some embodiments, the pH of one or more wash 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 a loading solution containing pro-rVWF, mat-rVWF/rVWF-PP complex, mat-rVWF and/or rVWF propeptide (rVWF-PP) as specified 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 cause the dissociation of the mat-rVWF/rVWF-PP complex into solution in step (a) of the process to mat-rVWF and rVWF-PP, said dissociation occurring due to the destruction of the non-covalently bound mat-rVWF and rVWFPP. In some embodiments, the pH of the feed solution is increased from at least about 7.2 to about 7.8. In some loading solution embodiments, the pH of the loading solution is increased to at least about 7.6. In some embodiments, the pH of the feed solution is increased by adding basic amino acids. 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 from 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 adding basic amino acids. In some embodiments, the one or more wash buffers have a pH of at least 7. In some embodiments, the pH of the one or more elution buffers is increased from 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 adding basic amino acids. In some embodiments, one or more elution buffers have a pH of at least 7.

In some embodiments, one or more buffers (including wash and/or elution buffers) contain one or more chelating agents. In some embodiments, the elution buffer contains at least one chelating agent. The chelating agent may 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 NTC, 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, NTK, and EDDS. In some embodiments, the chelating agent is HTC. 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 at b) contain said one or more chelating agents, and have a pH of at least 7.

Any of the buffers (buffer systems) described herein can be selected from the group consisting of glycine, HEPES (4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid), Tris-HCl (Tris(hydroxymethyl)aminomethane), histidine, imidazole, citrate acetate, citrate, acetate, solvate, phosphate, tris-HCl, bis-tris, histidine, imidazole, arginine HCl, lysine HCl and 2-(Nmorpholino)ethanesulfonic acid as separate buffers 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), tris-HCl (tris(hydroxymethyl)-aminomethane), histidine, imidazole, citrate acetate, MES and 2-(N-morpholino)ethanesulfonic acid. In some embodiments, the buffer contains citrate, acetate, MES, HEPES, phosphate, Tris-HCl and/or bis-Tris. In some embodiments, the buffer contains glycine. In some embodiments, the buffer contains HEPES (4-(2-hydroxyethyl)-1piperazineethanesulfonic acid). In some embodiments, the buffer contains Tris-HCl (Tris(hydroxymethyl)-aminomethane). In some embodiments, the buffer contains histidine. In some embodiments, the buffer contains an imidazole. In some embodiments, the buffer contains acetate citrate. In some embodiments, the buffer contains citrate. In some embodiments, the buffer contains acetate. In some embodiments, the buffer contains MES. In some embodiments, the buffer contains HEPES. In some embodiments, the buffer contains phosphate. In some embodiments, the buffer contains trisHCl. In some embodiments, the buffer contains bis-Tris. In some embodiments, the buffer contains histidine. In some embodiments, the buffer contains an imidazole. In some embodiments, the buffer contains arginine HCl. In some embodiments, the buffer contains lysine HCl. In some embodiments, the buffer contains 2-(Nmorpholino)ethanesulfonic acid. In some embodiments, the buffer comprises one, two, three, or four of the buffers described herein.

In some embodiments, one or more buffers (including wash and/or elution buffers) contain sodium citrate in a range including, but not limited to, 10 mM-80 mM sodium citrate, 15 mM-80 mM sodium citrate, 10 mM-80 mM citrate sodium, 15 mM-60 mM 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, or the like.

In some embodiments, the first elution buffer further contains sodium citrate in a range including, but not limited to, 10 mM-60 mM sodium citrate, 15 mM-60 mM sodium citrate, 10 mM-50 mM sodium citrate, 15 mM-50 mM 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, 60 mM sodium citrate, or the like.

In some embodiments, the second elution buffer further contains sodium citrate, such as, but not limited to, 10 mM-60 mM sodium citrate, 15 mM-60 mM sodium citrate, 10 mM-50 mM sodium citrate, 15 mM-50 mM 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, 60 mM sodium citrate, or the like.

In some embodiments, one or more buffers (including wash and/or elution buffers) in the cation exchange chromatography step contain EDTA while the desired rVWF species remain bound to the cation exchange resin. In some embodiments, one or more buffers (including wash and/or elution buffers) in the cation exchange chromatography step contain from about 0.5 mM to about 20 mM EDTA, for example, from about 0.5 mM to about 20 mM, from about 1 mM to about 20 mM, from about 1.5 mM to about 20 mM, from about 2 mM to about 20 mM, from about 3 mM to about 20 mM, from about 5 mM to about 20 mM, from about 0.5 mM to about 15 mM, from about 1 mM to about 10 mM, from about 1 mM to about 5 mM, about 5 mM, about 0.5 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM , about 18 mM, about 19 mM, about 20 mM, etc., as long as the desired rVWF species remains bound to the cation exchange resin. In some embodiments, buffers containing EDTA are used as part of a stepwise cation exchange elution. In some embodiments, buffers containing EDTA are used as part of a cation exchange gradient elution. In some embodiments, when EDTA is used as part of the buffers used in stepwise

- 20 045553 cation exchange elution, the counterion is Na ⁺ . In some embodiments, when EDTA is used as part of the buffers used in cation exchange gradient elutions, the counterion is Na ⁺ .

In some embodiments, citrate may be detected in the eluent after removal of the rVWF propeptide using a cation exchange elution method. In some embodiments, citrate may be detected in the eluent after removal of the rVWF propeptide using a stepwise cation exchange elution method. In some embodiments, citrate may be detected in the eluent after removal of the rVWF propeptide using a cation exchange gradient elution method. In some embodiments, the cation exchange counterion is Na ⁺ citrate.

In some embodiments, the conductivity of the buffers (including wash and/or elution buffers) is between 5 and 40 mS/cm, e.g., 5 mS/cm-40 mS/cm, 10 mS/cm-40 mS/cm, 15 mS/cm -40 mS/cm, 20 mS/cm-40 mS/cm, 5 mS/cm-15 mS/cm, 10 mS/cm-25 mS/cm, 15 mS/cm-30 mS/cm, 20 mS/cm -30 mS/cm or 30 mS/cm-40 mS/cm.

In some embodiments, the conductivity of the at least one wash buffer is from about 5 to about 40 mS/cm, for example, from about 5 to about 40 mS/cm, from about 10 to about 40 mS/cm, from about 15 to about 40 mS/cm, about 20 to about 40 mS/cm, about 25 to about 40 mS/cm, about 30 to about 40 mS/cm, about 10 to about 40 mS/cm, about 10 to about 30 mS/cm, about 5 to about 13 mS/cm, about 5 to about 15 mS/cm, about 15 to about 30 mS/cm, about 18 to about 40 mS/cm, or about 20 to about 40 mS/cm In other embodiments, the conductivity of the two or more wash buffers is from about 5 to about 40 mS/cm, for example, from about 5 to about 40 mS/cm, from about 10 to about 40 mS/cm, from about 15 to about 40 mS /cm, from about 20 to about 40 mS/cm, from about 25 to about 40 mS/cm, from about 30 to about 40 mS/cm, from about 10 to about 40 mS/cm, from about 10 to about 30 mS /cm, about 5 to about 13 mS/cm, about 5 to about 15 mS/cm, about 15 to about 30 mS/cm, about 18 to about 40 mS/cm, or about 20 to about 40 mS /cm.

In some embodiments, the conductivity of the at least one elution buffer is from about 5 to about 40 mS/cm, for example, from about 5 to about 40 mS/cm, from about 10 to about 40 mS/cm, from about 15 to about 40 mS/cm, about 20 to about 40 mS/cm, about 25 to about 40 mS/cm, about 30 to about 40 mS/cm, about 10 to about 40 mS/cm, about 10 to about 30 mS/cm, about 5 to about 13 mS/cm, about 5 to about 15 mS/cm, about 15 to about 30 mS/cm, about 18 to about 40 mS/cm, or about 20 to about 40 mS/cm In other embodiments, the conductivity of the two or more wash buffers is from about 5 to about 40 mS/cm, for example, from about 5 to about 40 mS/cm, from about 10 to about 40 mS/cm, from about 15 to about 40 mS /cm, from about 20 to about 40 mS/cm, from about 25 to about 40 mS/cm, from about 30 to about 40 mS/cm, from about 10 to about 40 mS/cm, from about 10 to about 30 mS /cm, about 5 to about 13 mS/cm, about 5 to about 15 mS/cm, about 15 to about 30 mS/cm, about 18 to about 40 mS/cm, or about 20 to about 40 mS /cm.

In some embodiments, the pH of the wash buffer is between pH 6.0 and pH 9.0, e.g., pH 6.0-pH 9.0, pH 6.3-pH 9.0, pH 6.5-pH 9.0 , pH 7.0-pH 9.0, pH 7.5-pH 9.0, pH 7.7.0-pH 9.0, pH 8.0-pH 9.0, pH 6.0-pH 8 .5, pH 6.5-pH 8.5, pH 7.0-pH 8.5, pH 7.5-pH 8.5, pH 6.0-pH 8.0, pH 6.5-pH 8 ,0, pH

7.0-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.

In one aspect, the method described herein includes a gradient elution step. The gradient elution step can remove product impurities and process-related impurities to optimize mature VWF yield. In some cases, the gradient elution step separates a higher percentage of VWF propeptide from mature VWF compared to the prior art method.

In some embodiments, the conductivity of one or more elution buffers is from about 5 to about 40 mS/cm, for example, from about 5 to about 40 mS/cm, from about 10 to about 40 mS/cm, from about 15 to about 40 mS /cm, from about 20 to about 40 mS/cm, from about 25 to about 40 mS/cm, from about 30 to about 40 mS/cm, from about 10 to about 40 mS/cm, from about 10 to about 30 mS /cm, about 5 to about 13 mS/cm, about 5 to about 15 mS/cm, about 15 to about 30 mS/cm, about 18 to about 40 mS/cm, or about 20 to about 40 mS /cm. In other embodiments, the conductivity of the two or more wash buffers is from about 5 to about 40 mS/cm, such as from about 5 to about 40 mS/cm, from about 10 to about 40 mS/cm, from about 15 to about 40 mS/cm /cm, from about 20 to about 40 mS/cm, from about 25 to about 40 mS/cm, from about 30 to about 40 mS/cm, from about 10 to about 40 mS/cm, from about to about 30 mS/ cm, about 5 to about 13 mS/cm, about 5 to about 15 mS/cm, about 15 to about 30 mS/cm, about 18 to about 40 mS/cm, or about 20 to about 40 mS/cm cm.

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

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

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

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 contain one or more non-reducing sugars. In some embodiments, the non-reducing sugar includes, but is not limited to, sucrose, trehalose, mannitol, sorbitol, galactitol, and/or xylitol. In some embodiments, the one or more buffers further contain one or more sugar alcohols. In some embodiments, the one or more buffers further comprise one or more polyols. In some embodiments, the sugar alcohol or polyol includes, without limitation, mannitol, xylitol, erythritol, threitol, sorbitol and/or glycerol. In some embodiments, the buffers further comprise sorbitol, mannitol, xylitol, sucrose, trehalose, ethylene glycol, propylene glycol, glycerol, 1,2,3-propanetriol, meso-erythritol and/or erythritol (meso-1,2,3,4-butantetrol ).

The pH of any of the buffers can be adjusted (increased) by adding amino acid, Tris, NaOH, ethanolamine, etc.

Any of the buffers (buffer systems) described herein may be selected from the group consisting of citrate, acetate, MES, HEPES, phosphate, Tris-HCl, bis-Tris, as individual buffers or as a combination of two or more buffers. In some embodiments, the buffer contains glycine. In some embodiments, the buffer contains citrate. In some embodiments, the buffer contains acetate. In some embodiments, the buffer contains MES. In some embodiments, the buffer contains HEPES. In some embodiments, the buffer contains phosphate. In some embodiments, the buffer contains Tris-HCl. In some embodiments, the buffer contains bis-Tris. In some embodiments, the buffer comprises one, two, three, or four of the buffers described herein.

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

Purification by size exclusion chromatography

In one aspect of the present invention, mature VWF and VWF-PP are separated using size exclusion chromatography (SEC). In some cases, remaining host cell-derived impurities such as CHO host cell proteins, process-related contaminants such as recombinant furin and small molecule virus inactivation reagents, media compounds such as soy peptone, and other process-related contaminants products are removed from mature VWF.

In another aspect of this method, mature VWF is separated from VWF-PP, such as residual VWFPP or free VWF-PP, using size exclusion chromatography. For separation, the starting composition may have a low pH and 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 greater, for example, pH 7.0 to pH 9.0. For example, the gradient elution buffer may contain EDTA and have a pH of 8.5.

In some embodiments, this invention provides a method for producing a composite

- 22 045553 item containing highly purified mature recombinant rVWF, depleted of propeptides, (matrVWF of high purity), and the specified method includes the stages: (a) loading a solution containing pro-rVWF, mat-rVWF/rVWF-PP complex, mat-rVWF and /or rVWF propeptide (rVWF-PP), to a size exclusion column, wherein said pro-rVWF, mat-rVWF/rVWF-PP complex and mat-rVWF are bound to said size exclusion column; (b) washing said size exclusion column in step a) containing said bound pro-rVWF, mat-rVWF/rVWF-PP complex and mat-rVWF, with one or more wash buffers; (c) treating said column in b) containing bound pro-rVWF, the mat-rVWF/rVWF-PP and mat-rVWF complex, with furin, wherein said furin cleaves said pro-rVWF into mat-rVWF and rVWF-PP; (d) eluting said bound pro-rVWF, the matrVWF/rVWF-PP complex and mat-rVWF from the column in c) with an elution buffer, wherein said elution buffer induces dissociation of said rVWF-PP from mat-rVWF non-covalently bound to said rVWF-PP wherein said dissociation is induced by: (i) adding at least one chelating agent to said elution buffer, or (ii) increasing the pH of said elution buffer to a pH of at least 7; and (e) collecting said mat-rVWF separately from said rVWF-PP to produce a highly purified mat-rVWF composition, wherein said highly purified mat-rVWF composition contains at least 95% mature rVWF and less than 5% rVWF-PP.

In some embodiments, a) and b) are performed simultaneously in the same step. In some embodiments, the solution in a) contains a stream from an immunoaffinity purification method. In some embodiments, the solution in a) includes a flow-through solution from a monoclonal antibody column, wherein said monoclonal antibody is a FVIII monoclonal antibody. In some embodiments, the solution in a) is selected from the group consisting of a cell culture medium, an antibody column flow-through solution, and a buffered solution.

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 greater or, in some cases, a pH of 7.0 or greater.

In some embodiments, the loading concentration of pro-VWF is from about 90 IU/ml to about 270 IU/ml resin, for example, 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 270 IU/ml, from about 140 IU/ml to about 270 IU/ml, from about 150 IU/ml to about 270 IU/ml, from about 90 IU/ml to about 250 IU/ml, from about 100 IU/ml to about 250 IU/ml, from about 110 IU/ml to about 250 IU/ml, from about 120 IU/ml to about 250 IU/ml, from about 130 IU/ml to about 250 IU/ml, from about 130 IU/ml ml to about 250 IU/ml, from about 140 IU/ml to about 250 IU/ml, from about 150 IU/ml to about 250 IU/ml, from about 90 IU/ml to about 200 IU/ml, from about 100 IU/ml to about 200 IU/ml, from about 110 IU/ml to about 200 IU/ml, from about 120 IU/ml to about 200 IU/ml, from about 130 IU/ml to about 200 IU/ml, from about 130 IU/ml to about 200 IU/ml, from about 140 IU/ml to about 200 IU/ml, from about 150 IU/ml to about 200 IU/ml, from about 90 IU/ml to about 100 IU/ml , from about 100 IU/ml to about 150 IU/ml, from about 150 IU/ml to about 200 IU/ml, from about 200 IU/ml to about 250 IU/ml or from about 250 IU/ml to about 270 IU /ml resin.

In some embodiments, the pH of the feed composition, feed solution, or feed composition 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, for example, pH 6.0-pH 9.0, pH 6.3-pH 9.0, pH 6.5-pH 9.0, pH 7.0-pH 9.0, pH 7.5 -pH 9.0, pH 7.7, pH 9.0, pH 8.0, pH 9.0, pH 6.0, pH 8.5, pH 6.5, pH 8.5, pH 7 ,0-pH 8.5, pH 7.5-pH 8.5, pH 6.0-pH 8.0, pH 6.5-pH 8.0, pH 7.0-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 or pH 9.0.

In some embodiments, the size exclusion method includes a buffer system. In some embodiments, the buffer system includes one or more buffers for separation. In some embodiments, the buffer system includes at least one separation buffer. In some embodiments, the buffer system contains 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.

In some embodiments, the first separation buffer contains 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 buffer has a pH in the range of pH 6.0 to pH 9.0 and optionally contains 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 may contain at least one chelating agent and

- 23 045553 has a pH in the range from pH 6.0 to pH 6.9. In some embodiments, the first separation buffer has a pH of less than 7. In some embodiments, the second separation buffer has a pH of greater than 7. In some embodiments, when two separation buffers are used, the first separation buffer has a pH of less than 7 and the second separation buffer has a pH of less than 7. for separation it has a pH greater than 7.

In some embodiments, the stock solution containing mature rVWF and rVWF-PP is contacted with a separation buffer containing at least one chelating agent, and optionally a buffer having a pH in the range of pH 6.0 to pH 9.0. In some embodiments, the feed solution is contacted with a buffer having a pH in the range of pH 6.0 to pH 9.0, and optionally a buffer containing 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 with a pH of 6.0 to 6.9. In some embodiments, the buffer is a second separation buffer with a pH between 7.0 and 9.0. In some embodiments, the stock 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.

In some embodiments, the pH of the one or more separation buffers is between pH 6.0 and pH 9.0, e.g., pH 6.0-pH 9.0, pH 6.3-pH 9.0, pH 6.5- pH 9.0, pH 7.0-pH 9.0, pH 7.5-pH 9.0, pH 7.7.0-pH 9.0, pH 8.0-pH 9.0, pH 6, 0-pH 8.5, pH 6.5-pH 8.5, pH 7.0-pH 8.5, pH 7.5-pH 8.5, pH 6.0-pH 8.0, pH 6, 5-pH 8.0, pH 7.0-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.

In some embodiments, the pH of the elution buffer is increased compared to the initial solution in step a), increased compared to the first separation buffer when two separation buffers are used, and/or increased compared to the first separation buffer when a second separation buffer is used. divisions. In some embodiments, when a first separation buffer and a second separation buffer are used, the first separation buffer has a pH of less than 7 and the second separation buffer has a pH of greater than 7.

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 compared to a loading solution containing pro-rVWF, mat-rVWF/rVWF-PP complex, mat-rVWF and/or rVWF propeptide (rVWF-PP) as specified in method step (a). 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 cause the matrVWF/rVWF-PP complex to dissociate into solution in step (a) of the process to mat-rVWF and rVWF-PP, said dissociation occurring due to the destruction of the non-covalently bound mat-rVWF and rVWF-PP. In some embodiments, the pH of the feed solution is increased from at least about 7.2 to about 7.8. In some loading solution embodiments, the pH of the loading solution is increased to at least about 7.6. In some embodiments, the pH of the feed solution is increased by adding basic amino acids. 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 from 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 adding basic amino acids. In some embodiments, the one or more separation buffers have a pH of at least 7.

In some embodiments, the one or more separation buffers contain one or more chelating agents. In some embodiments, the elution buffer contains at least one chelating agent. The chelating agent may 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 NTC, DTPA, EDDS, EDTA, EGTA, CDTA, and citrate. In some embodiments, the chelating agent is HTC. In some embodiments, the chelating agent is DTPC. 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 at b) contain said one or more chelating agents, and have a pH of at least 7.

In some embodiments, the one or more separation buffers contain at least one chelating agent. The chelating agent may be a chelating agent

- 24 045553 agent with a divalent cation. In some embodiments, the divalent cation chelating agent is selected from the group consisting of nitrile-2,2',2-triacetic acid (NTA), diethylenetriaminepentaacetic acid; diethylenetriamine-K,K,K',K',K-pentaacetic acid (DTPA), ethylenediamine-K,K'-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 NTK, DTPA, EDDS, EDTA, and citrate. In some embodiments, the chelating agent is HTC. In some embodiments, the chelating agent is DTPC. 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, elution buffer A and/or elution buffer B in the anion exchange chromatography step contains from about 0.5 mM to about 20 mM EDTA, for example, from about 0.5 mM to about 20 mM, from about 1 mM to about 20 mM, about 1.5 mM to about 20 mM, about 2 mM to about 20 mM, about 3 mM to about 20 mM, about 5 mM to about 20 mM, about 0.5 mM to about 15 mM , about 1 mM to about 10 mM, about 1 mM to about 5 mM, about 5 mM, about 0.5 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM , about 19 mM, about 20 mM, or the like.

In some embodiments, one or more separation buffers contain sodium citrate in a range including, but not limited to, 10 mM-500 mM sodium citrate, 15 mM-400 mM sodium citrate, 10 mM-400 mM sodium citrate, 15 mM-350 mM sodium citrate, 20 mM-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, 90 mM sodium citrate, 100 mM sodium citrate, 110 mM sodium citrate, 120 mM sodium citrate, 130 mM sodium citrate, 140 mM sodium citrate, 150 mM sodium citrate , 160 mM sodium citrate, 170 mM sodium citrate, 180 mM sodium citrate, 190 mM sodium citrate, 200 mM sodium citrate, 210 mM sodium citrate, 220 mM sodium citrate, 230 mM sodium citrate, 240 mM sodium citrate, 25 0 mM sodium citrate , 260 mM sodium citrate, 270 mM sodium citrate, 280 mM sodium citrate, 290 mM sodium citrate, 300 mM sodium citrate, 310 mM sodium citrate, 320 mM sodium citrate, 330 mM sodium citrate, 340 mM sodium citrate, 35 0 mM sodium citrate , 360 mM sodium citrate, 370 mM sodium citrate, 380 mM sodium citrate, 390 mM sodium citrate, 400 mM sodium citrate, 410 mM sodium citrate, 420 mM sodium citrate, 430 mM sodium citrate, 440 mM sodium citrate, 45 0 mM sodium citrate , 460 mM sodium citrate, 470 mM sodium citrate, 480 mM sodium citrate, 490 mM sodium citrate, 500 mM sodium citrate, 510 mM sodium citrate, 520 mM sodium citrate, 530 mM sodium citrate, 540 mM sodium citrate, 55 0 mM sodium citrate , 560 mM sodium citrate, 570 mM sodium citrate, 580 mM sodium citrate, 590 mM sodium citrate or 600 mM sodium citrate, or the like.

In some embodiments, the one or more separation buffers further contain sodium citrate in a range including, but not limited to, 10 mM-500 mM sodium citrate, 15 mM-400 mM sodium citrate, 10 mM-400 mM sodium citrate, 15 mM-350 mm sodium citrate, 20 mm-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 of citrate sodium, 70 mM sodium citrate, 75 mM sodium citrate, 80 mM sodium citrate, 90 mM sodium citrate, 100 mM sodium citrate, 110 mM sodium citrate, 120 mM sodium citrate, 130 mM sodium citrate, 140 mM sodium citrate, 150 mM citrate sodium, 160 mM sodium citrate, 170 mM sodium citrate, 180 mM sodium citrate, 190 mM sodium citrate, 200 mM sodium citrate, 210 mM sodium citrate, 220 mM sodium citrate, 230 mM sodium citrate, 240 mM sodium citrate, 25 0 mM citrate sodium, 260 mM sodium citrate, 270 mM sodium citrate, 280 mM sodium citrate, 290 mM sodium citrate, 300 mM sodium citrate, 310 mM sodium citrate, 320 mM sodium citrate, 330 mM sodium citrate, 340 mM sodium citrate, 35 0 mM citrate sodium, 360 mM sodium citrate, 370 mM sodium citrate, 380 mM sodium citrate, 390 mM sodium citrate, 400 mM sodium citrate, 410 mM sodium citrate, 420 mM sodium citrate, 430 mM sodium citrate, 440 mM sodium citrate, 45 0 mM citrate sodium, 460 mM sodium citrate, 470 mM sodium citrate, 480 mM sodium citrate, 490 mM sodium citrate, 500 mM sodium citrate, 510 mM sodium citrate, 520 mM sodium citrate, 530 mM sodium citrate, 540 mM sodium citrate, 55 0 mM citrate sodium, 560 mM sodium citrate, 570 mM sodium citrate, 580 mM sodium citrate, 590 mM sodium citrate or 600 mM sodium citrate, or the like.

In some embodiments, the one or more separation buffers further contain sodium citrate, such as, but not limited to, 10 mM-500 mM sodium citrate, 15 mM-400 mM sodium citrate, 10 mM-400 mM sodium citrate, 15 mM-350 mM sodium citrate, 20 mM-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, 90 mM sodium citrate, 100 mM sodium citrate, 110 mM

- 25 045553 sodium citrate, 120 mM sodium citrate, 130 mM sodium citrate, 140 mM sodium citrate, 150 mM sodium citrate, 160 mM sodium citrate, 170 mM sodium citrate, 180 mM sodium citrate, 190 mM sodium citrate, 200 mM sodium citrate , 210 mM sodium citrate, 220 mM sodium citrate, 230 mM sodium citrate, 240 mM sodium citrate, 250 mM sodium citrate, 260 mM sodium citrate, 270 mM sodium citrate, 280 mM sodium citrate, 290 mM sodium citrate, 30 0 mM sodium citrate , 310 mM sodium citrate, 320 mM sodium citrate, 330 mM sodium citrate, 340 mM sodium citrate, 350 mM sodium citrate, 360 mM sodium citrate, 370 mM sodium citrate, 380 mM sodium citrate, 390 mM sodium citrate, 40 0 mM sodium citrate , 410 mM sodium citrate, 420 mM sodium citrate, 430 mM sodium citrate, 440 mM sodium citrate, 450 mM sodium citrate, 460 mM sodium citrate, 470 mM sodium citrate, 480 mM sodium citrate, 490 mM sodium citrate, 50 0 mM sodium citrate , 510 mM sodium citrate, 520 mM sodium citrate, 530 mM sodium citrate, 540 mM sodium citrate, 550 mM sodium citrate, 560 mM sodium citrate, 570 mM sodium citrate, 580 mM sodium citrate, 590 mM sodium citrate or 60 0 mM sodium citrate , or the like

In some embodiments, the conductivity of the separation buffer is from about 5 mS/cm to about 40 mS/cm, for example, 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 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, 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.

Any of the buffers (buffer systems) described herein 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, as individual buffers or as a combination of two or more buffers. In some embodiments, the buffer contains glycine. In some embodiments, the buffer includes 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 contains citrate. In some embodiments, the buffer contains acetate. In some embodiments, the buffer contains MES. In some embodiments, the buffer contains HEPES. In some embodiments, the buffer contains phosphate. In some embodiments, the buffer contains Tris-HCl. In some embodiments, the buffer contains bis-Tris.

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

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

In some embodiments, the flow rate of one or more separation buffer stages of the method is from about 10 cm/hour to about 200 cm/hour, for example, about 10 cm/hour, about 15 cm/hour, about 20 cm/hour , about 25 cm/h, about 30 cm/h, about 35 cm/h, about 40 cm/h, about 45 cm/h, about 50 cm/h, about 55 cm/h, about 60 cm/h, about 65 cm/h, about 70 cm/h, about 75 cm/h, about 80 cm/h, about 85 cm/h, about 90 cm/h, about 95 cm/h, about 100 cm/h, about 105 cm /h, about 110 cm/h, about 115 cm/h, about 120 cm/h, about 125 cm/h, about 130 cm/h, about 135 cm/h, about 140 cm/h, about 145 cm/h , about 150 cm/h, about 155 cm/h, about 160 cm/h, about 165 cm/h, about 170 cm/h, about 175 cm/h, about 180 cm/h, about 185 cm/h, about 190 cm/h, about 195 cm/h or about 200 cm/h. Depending on the resin, in some embodiments the flow rate can be as high as 600 cm/h.

In some embodiments, the flow rate of one or more separation buffer stages of the method is from about 10 cm/hour to about 200 cm/hour, for example, about 10 cm/hour, about 15 cm/hour, about 20 cm/hour , about 25 cm/h, about 30 cm/h, about 35 cm/h, about 40 cm/h, about 45 cm/h, about 50 cm/h, about 55 cm/h, about 60 cm/h, about 65 cm/h, about 70 cm/h, about 75 cm/h, about 80 cm/h, about 85 cm/h, about 90 cm/h, about 95 cm/h, about 100 cm/h, about 105 cm /h, about 110 cm/h, about 115 cm/h, about 120 cm/h, about 125 cm/h, about 130 cm/h, about 135 cm/h, about 140 cm/h, about 145 cm/h ,

- 26 045553 about 150 cm/h, about 155 cm/h, about 160 cm/h, about 165 cm/h, about 170 cm/h, about 175 cm/h, about 180 cm/h, about 185 cm/h , about 190 cm/h, about 195 cm/h or about 200 cm/h. Depending on the resin, in some embodiments the flow rate can be as high as 600 cm/h.

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 contain one or more non-reducing sugars. In some embodiments, the non-reducing sugar includes, but is not limited to, sucrose, trehalose, mannitol, sorbitol, galactitol, and/or xylitol. In some embodiments, the one or more buffers further contain one or more sugar alcohols. In some embodiments, the one or more buffers further comprise one or more polyols. In some embodiments, the sugar alcohol or polyol includes, without limitation, mannitol, xylitol, erythritol, threitol, sorbitol and/or glycerol. In some embodiments, the buffers further comprise sorbitol, mannitol, xylitol, sucrose, trehalose, ethylene glycol, propylene glycol, glycerol, 1,2,3-propanetriol, meso-erythritol and/or erythritol (meso-1,2,3,4-butantetrol ).

In some embodiments, the combination of buffer chelators for the size exclusion chromatography method includes citrate, malate (malic acid), and tartrate (tartaric acid).

Immunoaffinity purification

In some embodiments, a solution containing the pro-rVWF complex, mat-rVWF/rVWFPP, mat-rVWF, and/or rVWF propeptide (rVWF-PP) is prepared by immunoaffinity purification, including, for example, 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 in the art and have been described. 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 an anti-VWF monoclonal antibody and Factor VIII is dissociated from the complex by CaCl ions. ₂ . The immunoaffinity support, on which vWF is still adsorbed, is regenerated with a chaotropic agent, in particular NaSCN, with a vWF/NaSCN solution formed as a by-product and discarded.

Other methods include those described in US Pat. No. 6,579,723, also incorporated herein by reference in its entirety, which describes a method for isolating highly purified vWF or factor VIII/vWF complex using an immunoaffinity chromatography procedure. This method involves the recovery of VWF from an immunoaffinity adsorbent using an elution agent containing zwitterionic particles. The presence of zwitterionic particles allows the use of mild conditions throughout production, helping to maintain the molecular integrity, activity and incorporation of reduced proteins into pharmaceutical formulations without the need for additional stabilizers or preservatives.

Any such methods can be used with the current purification method to obtain a solution containing pro-rVWF, mat-rVWF/rVWF-PP complex, mat-rVWF and/or rVWF propeptide (rVWF-PP). In some embodiments, immunoaffinity purification optionally occurs prior to step (a) in any of the purification procedures described herein, including those based on cation exchange, anion exchange, and/or size exclusion chromatography procedures.

Free mature VWF

In some embodiments, the level of host cell impurities (HC) in the composition provided herein is equal to or less than 2.0 ppm, e.g., 2.0 ppm, 1.9 ppm. /million, 1.8 ppm, 1.7 ppm, 1.6 ppm, 1.5 ppm, 1.4 ppm, 1 .3 ppm, 1.2 ppm, 1.1 ppm, 1.0 ppm, 0.9 ppm, 0.8 ppm million, 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 level of host cell impurities in the composition provided herein is equal to or less than 0.6 ppm, for example, 0.6 ppm, 0.5 ppm. , 0.4 ppm, 0.3 ppm, 0.2 ppm, 0.1 ppm, 0.09 ppm, 0.08 h ./million, 0.07 ppm, 0.06 ppm, 0.05 ppm, 0.04 ppm, 0.03 ppm, 0.02 ppm, 0.01 ppm or less.

In some embodiments, the level of host cell impurities (HC) in a composition provided herein is equal to or less than 5.0% (eg, <5.0%). In some embodiments, the level of host cell impurities (HC) in a composition provided herein is equal to or less than 4.0% (eg, <4.0%). In some embodiments, the level of host cell impurities (HC) in a composition provided herein is equal to or less than 3.0% (eg, <3.0%). In some embodiments, the level of host cell impurities (HC) in a composition provided herein is equal to or less than 2.0% (eg, <1.0%). In some embodiments, the level of host cell impurities (HC) in the composition provided herein is equal to or less than 2.0%

- 27 045553 (for example, <1.0%). In some embodiments, the level of host cell impurities (HC) is equal to or less than 0.9% (eg, <0.9%). In some embodiments, the level of host cell impurities (HC) is equal to or less than 0.8% (eg, <0.8%). In some embodiments, the level of host cell impurities (HC) is equal to or less than 0.7% (eg, <0.7%). In some embodiments, the level of host cell impurities (HC) is equal to or less than 0.6% (eg, <0.6%). In some embodiments, the level of host cell impurities (HC) is equal to or less than 0.5% (eg, <0.5%). In some embodiments, the level of host cell impurities (HC) is equal to or less than 0.4% (eg, <0.4%). In some embodiments, the level of host cell impurities (HC) is equal to or less than 0.3% (eg, <0.3%). In some embodiments, the level of host cell impurities (HC) is equal to or less than 0.2% (eg, <0.2%). In some embodiments, the level of host cell impurities (HC) is equal to or less than 0.1% (eg, <0.1%).

In some embodiments, the rVWF-PP impurity is 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 0.3%, less than 0.2%, less than 0.1% or less than 0.05%. In some embodiments, the rVWF-PP impurity is less than 15%. In some embodiments, the rVWF-PP impurity is less than 10%. In some embodiments, the rVWF-PP impurity is less than 5%. In some embodiments, the rVWF-PP impurity is less than 4%. In some embodiments, the rVWF-PP impurity is less than 3%. In some embodiments, the rVWF-PP impurity is less than 2%. In some embodiments, the rVWF-PP impurity is less than 1%. In some embodiments, the rVWF-PP impurity is less than 0.5%. In some embodiments, the rVWF-PP impurity is less than 0.4%. In some embodiments, the rVWF-PP impurity is less than 0.3%. In some embodiments, the rVWF-PP impurity is less than 0.2%. In some embodiments, the rVWF-PP impurity is less than 0.1%. In some embodiments, the rVWF-PP impurity is less than 0.05%.

Table 1

Exemplary removal capacity of VWF-PP

Stud AND I Loading, VWF-PP admixture Eluate, VWF-PP admixture % (mass/mass) % (mass/mass) AEX -thirty% * -12% SEX -thirty% ~<0.1% SEC -12% ~<0.1% *: either pre-ripened before loading, or ripened to completion by in vitro column ripening (as currently carried out in the process and part of the claims of another patent)

Production of recombinant VWF

The free mature recombinant von Willebrand factor (rVWF) of the present invention can be produced recombinantly. One skilled in the art will recognize suitable methods for expressing a recombinant protein in a host cell. In some cases, the method includes expressing a nucleic acid sequence encoding rVWF in a host cell, such as a CHO cell, and culturing the resulting host cell under certain conditions to produce rVWF, prepro-VWF, pro-VWF, and the like.

In some embodiments, the nucleic acid sequence comprising the VWF coding sequence may be an expression vector. The vector may be delivered by a virus or may be a plasmid. The nucleic acid sequence encoding the protein may 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 may further comprise other sequences suitable for controlled protein expression, such as promoter sequences, enhancers, TATA boxes, transcription start sites, polylinkers, restriction sites, poly-A sequences, protein processing sequences, selection markers, and the like, which are generally known to one skilled in the art.

A wide variety of vectors can be used to express VWF, which can be selected from eukaryotic expression vectors. Examples of eukaryotic expression vectors include: (i) yeast expression vectors such as pAO, pPIC, pYES, pMET, using

- 28 045553 promoters such as AOX1, GAP, GAL1, AUG1, etc.; (ii) vectors for expression in insect cells such as pMT, pAc5, pIB, pMIB, pBAC, etc., using promoters such as PH, p10, MT, Ac5, OpIE2, gp64, polh, etc. ., and (iii) vectors for expression in mammalian cells, such as pSVL, pCMV, pRc/RSV, pcDNA3, pBPV, etc., and vectors derived from viral systems such as vaccinia virus, adeno-associated viruses, herpes viruses , retroviruses, etc., using promoters such as CMV, SV40, EF-1, UbC, RSV, ADV, BPV and β-actin.

In some aspects, rVWF used in the methods of this invention is produced by expression in cultured mammalian cells using methods known in the art. In specific embodiments, the mammalian culture contains CHO cells. In additional embodiments, rVWF is coexpressed with recombinant factor VIII (rFVIII) in the same culture. In such embodiments, rVWF and rFVIII are purified together (co-purified) or separately using methods known in the art. In other embodiments, rVWF is expressed in a culture that does not contain rFVIII.

In some embodiments, rVWF is expressed and isolated from a suitable eukaryotic host system. Examples of eukaryotic cells include, but are not limited to, mammalian cells such as CHO, COS, HEK 293, BHK, SK-Hep and HepG2; insect cells, such as SF9 cells, SF21 cells, S2 cells and High Five cells; and yeast cells, such as Saccharomyces cells or

Schizosaccharomyces. In one embodiment, VWF can be expressed in yeast cells, insect cells, avian cells, mammalian cells, and the like. For example, in a human cell line, a hamster cell line, or a mouse cell line. In one specific embodiment, the cell line is a CHO, BHK, or HEK cell line. Typically, mammalian cells, for example, CHO cells from a continuous cell line, can be used to express the VWF of the present invention. In certain cases, the VWF protein is expressed and released from the CHO cell expression system.

VWF can be produced in a cell culture system or any cell culture method of those skilled in the art. In some embodiments, cell culture can be performed in large bioreactors under conditions suitable to provide large volumetric surface areas of the culture to achieve high cell densities and protein expression. One means of providing such growth conditions is the use of microcarriers for culturing cells in stirred tank bioreactors. The concept of cell growth on microcarriers was first described by van Wezel (van Wezel, A. L., Nature, 1967, 216:64-5) and allows cells to be attached to the surface of small solid particles suspended in the growth medium. These methods provide a high surface to volume ratio and thus allow efficient use of nutrients. Additionally, for the expression of secreted proteins in eukaryotic cell lines, an increased surface to volume ratio allows for higher levels of secretion and therefore higher yields of proteins in the culture supernatant. Finally, these methods allow easy expansion of eukaryotic expression cultures.

Cells expressing VWF can be associated with a spherical or porous microcarrier during cell culture growth. The microcarrier may be a microcarrier selected from the group of microcarriers based on dextran, collagen, plastic, gelatin and cellulose and others, as described in Butler (1988. In: Spier & Griffiths, Animal Cell Biotechnology 3:283-303). It is also possible to grow cells to biomass on spherical microcarriers and subculture the cells when they reach final fermenter biomass and to production of expressed protein on a porous microcarrier, or vice versa. Suitable spherical microcarriers may include smooth surface microcarriers such as Cytodex™ 1, Cytodex™ 2 and Cytodex™ 3 (GE Healthcare) and macroporous microcarriers such as Cytopore™ 1, Cytopore™ 2, Cytoline™ 1 and Cytoline™ 2 ( GE Healthcare).

In another embodiment, the VWF propeptide is cleaved from immature VWF in vitro by exposing the pro-VWF to furin. In some embodiments, the furin used to cleave the propeptide is a recombinant furin.

In some embodiments, rVWF is expressed in cells cultured in a cell culture medium that produces high molecular weight rVWF. The terms cell culture solution, cell culture medium or media, and cell culture supernatant refer to aspects of cell culture processes generally well known in the art. In the context of the present invention, the cell culture solution may comprise a cell culture medium and a cell culture supernatant. Cell culture medium is added externally to the cell culture solution, optionally along with additives, to provide nutrients and other components for culturing cells expressing VWF. Cell culture supernatant refers to a cell culture solution containing nutrients and other components from the cell culture medium, as well as products released, metabolized and/or excreted from the cells during culture. In to

- 29 045553 In additional embodiments, the medium may not contain animal proteins and have a certain chemical composition. Methods for preparing a chemically defined culture medium that does not contain animal proteins are known in the art, for example in US 2006/0094104, US 2007/0212770 and US 2008/0009040, which are both incorporated herein for all purposes and in particular , for all objects related to cell culture media. Protein-free and related terms refer to a protein that comes from a source exogenous to or different from the cells in culture that naturally secrete proteins during growth. In another embodiment, the culture medium does not contain polypeptides. In another embodiment, the culture medium does not contain serum. In another embodiment, the culture medium does not contain animal proteins. In another embodiment, the culture medium does not contain components of animal origin. In another embodiment, the culture medium contains a protein, such as animal protein from whey, such as fetal calf serum. In another embodiment, recombinant proteins are added exogenously to the culture. In another embodiment, the proteins are obtained from a certified pathogen-free animal. As used herein, the term chemically defined means that the medium does not contain any unspecified additives such as, for example, extracts of animal, organ, glandular, plant or yeast components. Accordingly, each component of a chemically defined environment is precisely defined. In a preferred embodiment, the media is free of animal components and proteins.

In some embodiments, the VWF-expressing cell culture may 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 longer. The cell density at which cell culture is maintained to produce recombinant VWF protein will depend on the culture conditions and the medium used to express the protein. One skilled in the art will readily be able to determine the optimal cell density for a VWF-producing cell culture. In one embodiment, the culture is maintained at a cell density of about 0.5x106 to ^4x107 cells/ml for an extended period of time. In other embodiments, the cell density is maintained at a concentration of from about 1.0 x 10 6 to about 1.0 x 10 ⁷ cells/ml for an extended period of time. In other embodiments, the cell density is maintained at a concentration of from about 1.0x106 to about 4.0x106 cells/ml for an extended period of time. In other embodiments, the cell density is maintained at a concentration of from about 1.0x106 to about 4.0x106 cells/ml for an extended period of time. In other embodiments, the cell density may be maintained at about 2.0x106 to about 4.0x106, or about 1.0x106 to about 2.5x106, or about 1.5x106 to about 3.5x106, or any other similar range over a long period of time. After suitable time in cell culture, rVWF can be isolated from the expression system using methods known in the art.

In a specific embodiment, the cell density in the continuous cell culture for producing rVWF is maintained at a level of no more than 2.5 x 106 cells/ml for an extended period. In other specific embodiments, the cell density is maintained at no more than 2.0 x 10 cells/mL, 1.5 x 10 6 cells/mL, 1.0 x 10 6 cells/mL, 0.5 x 10 6 cells/mL or less. In one embodiment, the cell density is maintained at a level between 1.5 x 106 cells/ml and 2.5 x 106 cells/ml.

In one embodiment of the cell cultures described above, the cell culture solution contains a media supplement containing copper. Such cell culture solutions are described, for example, in US Pat. No. 8,852,888 and US Pat. No. 9,409,971, which are incorporated herein by reference in their entirety for all purposes and, in particular, for all provisions relating to cell culture methods and compositions for the production of recombinant VWF.

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

One form of beneficial rVWF has at least in vivo stabilizing property, e.g.

- 30 045553 binding to at least one factor VIII (FVIII) molecule and possibly having a pharmacologically acceptable glycosylation pattern. Specific examples of this include VWF without the A2 domain, so it is resistant to proteolysis (Lankhof et al., Thromb. Haemost. 77: 1008-1013, 1997) and the VWF fragment from Val 449 to Asn 730, including the glycoprotein 1b-binding domain and sites binding for collagen and heparin (Pietu et al., Biochem. Biophys. Res. Commun. 164: 1339-1347, 1989). Determination of the ability of VWF to stabilize at least one FVIII molecule, in one aspect, is carried out in mammals with VWF deficiency in accordance with methods known in the art.

The RVWF of this invention can be prepared by any method known in the art. One specific example is disclosed in WO 86/06096, published October 23, 1986, and US Patent Application No. 07/559509, filed July 23, 1990, which is incorporated herein by reference with respect to methods for producing recombinant VWF. Thus, methods are known in the art for (i) producing recombinant DNA by genetic engineering, for example by reverse transcription of RNA and/or DNA amplification, (ii) introducing recombinant DNA into prokaryotic or eukaryotic cells by transfection, for example by electroporation or microinjection, (iii) culturing the transformed cells, e.g., in a continuous or batch manner, (iv) expressing VWF, e.g., constitutively or by induction, and (v) isolating VWF, e.g., from the culture medium or by harvesting the transformed cells, so that (vi ) obtain purified rVWF, for example by anion exchange chromatography or affinity chromatography. Recombinant VWF, in one aspect, is produced in transformed host cells using recombinant DNA techniques well known in the art. For example, polypeptide coding sequences can be excised from DNA using suitable restriction enzymes. Alternatively, in another aspect, the DNA molecule is synthesized using chemical synthesis methods such as the phosphoramidate method. Moreover, in another aspect, a combination of these methods is used.

The present invention also provides vectors encoding the polypeptides of the invention in a suitable host. The vector includes a polynucleotide that encodes a polypeptide operably linked to appropriate expression control sequences. Methods for effecting this functional linkage before or after insertion of the polynucleotide into a 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 associated with the control of transcription or translation. The resulting vector containing the polynucleotide is used to transform a suitable host. This conversion can be performed using methods well known in the art.

Any of the large number of available and well known host cells are used in the practice of the present invention. The choice of a particular host depends on a number of factors known in the art, including, for example, compatibility with the selected expression vector, toxicity of the peptides encoded by the DNA molecule, transformation rate, ease of peptide isolation, expression characteristics, biosafety and cost. A balance of these factors must be achieved with the understanding that not all host cells are equally efficient at expressing a particular DNA sequence. For the purposes of these general guidelines, beneficial microbial host cells include, but are not limited to, bacteria, yeast and other fungi, insects, plants, mammalian (including human) cells in culture, or other hosts known in the art.

The transformed host cells are cultured under normal culture conditions so that the desired compounds are expressed. Such culture conditions are well known in the art. Finally, the polypeptides are purified from the culture medium or the host cells themselves by methods well known in the art.

Depending on the host cell used to express the compound of the invention, carbohydrate (oligosaccharide) groups are optionally attached to sites that are known to be glycosylation sites in proteins. In general, O-linked oligosaccharides are attached to serine (Ser) or threonine (Thr) residues, while N-linked oligosaccharides are attached to asparagine (Asn) residues when they are part of the Asn-X-Ser/Thr sequence, where X can be any amino acid except proline. X is preferably one of the 19 naturally occurring amino acids, excluding proline. The structures of N-linked and O-linked oligosaccharides and sugar residues found in each type are different. One type of sugar that is commonly found in both N-linked and O-linked oligosaccharides is N-acetylneuraminic acid (called sialic acid). Sialic acid is typically the terminal residue of both N-linked and O-linked oligosaccharides and, due to its negative charge, in one aspect imparts acidic properties to the glycosylated compound. Such site(s) may be included in a linker of the compounds of the present invention and are preferably glycosylated by the cell during recombinant production of polypeptide compounds (eg, in mammalian cells such as CHO, BHK, COS). In other aspects, such sites are glycosylated using synthetic or semi-synthetic procedures known in the art.

- 31 045553

In some embodiments, sialysis (also referred to as 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 results in increased stability of the rVWF compared to rVWF that has not been sialylated. In some embodiments, sialylation results in increased stability of rVWF in the bloodstream (eg, after administration to a subject) compared to rVWF that has not been sialylated. In some embodiments, the stability of sialylated rVWF is increased by 10, 20, 30, 40, 50, 60, 70, 80, 90% or more compared to rVWF that has not been sialylated. In some embodiments, sialylation results in an increase in the half-life of rVWF compared to rVWF that has not been sialylated. In some embodiments, sialylation results in an increase in the half-life of rVWF in the bloodstream (eg, after administration to a subject) compared to rVWF that has not been sialylated. In some embodiments, the half-life of sialylated rVWF is increased by 10, 20, 30, 40, 50, 60, 70, 80, 90% or more compared to rVWF that has not been sialylated. In some embodiments, the increased half-life of sialylated rVWF results in rVWF that is stable for 1, 2, 3, 4, 6, 12, 24 hours or more in the bloodstream (e.g., after administration to a subject) compared to rVWF that has not been exposed sialylation. In some embodiments, sialylation increases the amount of 2,3-sialylation and/or 2,6-sialylation. In some embodiments, sialylation is enhanced by adding 2,3 sialyltransferase and/or 2,6-sialyltransferase and CMP-NANA (cytidine-5'-monophosphoN-acetylneuraminic acid sodium salt) as an additional buffer step. In some embodiments, sialylation is enhanced by adding 2,3-sialyltransferase and CMP-NANA (cytidine-5'-monophospho-N-acetylneuraminic acid sodium salt) as an additional buffer step. In some embodiments, 2,3-sialylation is enhanced by adding 2,3-sialyltransferase and CMP-NANA (cytidine-5'-monophospho-N-acetylneuraminic acid sodium salt) as an additional buffer step. In some embodiments, to increase sialylation, the bound protein (eg, bound rVWF) is treated with a sialidase (eg, 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 2,6-sialyltransferase and CMP-NANA.

In some embodiments, 2,6-sialylation is enhanced by adding 2,6 sialyltransferase and CMP-NANA (cytidine-5'-monophospho-N-acetylneuraminic acid sodium salt) as an additional buffer step. In some embodiments, 2,3-sialylation and/or 2,6-sialylation is enhanced by adding 2,3-sialyltransferase and/or 2,6-sialyltransferase and CMP-NANA (cytidine-5'-monophospho-N-acetylneuraminic acid sodium salt) as additional buffer stage. In some embodiments, CMP-NANA is chemically or enzymatically modified to transfer the modified sialic acid to a potentially free position. In some embodiments, sialylation is performed by loading rVWF onto a resin, washing with one or more buffers as described herein to remove undesirable impurities, using one or more buffers containing sialyltransferase and CMP-NANA under conditions allowing for additional sialylation, and washing with one or more buffers to remove excess sialylation reagents, and eluting with one or more buffers of an enhanced rVWF (eg, enhanced sialylation rVWF). In some embodiments, the sialylation process is performed as part of a cation exchange method, anion exchange method, size exclusion method, or immunoaffinity purification method, as described herein.

Alternatively, the compounds are prepared synthetically using, for example, solid phase synthesis methods. Suitable methods 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. Pat. No. 3,941,763; Finn et al. (1976), The Proteins (3rd ed.) 2: 105253; and Erickson et al. (1976), The Proteins (3rd ed.) 2: 257-527'. Solid-phase synthesis is the preferred method for preparing individual peptides because it is the most cost-effective method for producing small peptides.

VWF fragments, variants and analogues can be prepared by methods well known in the art. Polypeptide fragments can be produced using, but not limited to, enzymatic digestion (eg, trypsin, chymotrypsin), as well as using recombinant means to create polypeptide fragments having a specific amino acid sequence. Polypeptide fragments can be prepared comprising a region of a protein having a particular activity, such as a multimerization domain or any other identifiable VWF domain known in the art.

Methods for preparing polypeptide analogues are also well known. Amino acid analogues

- 32 045553 polypeptide sequences can be analogues with substitution, insertion, addition or deletion. Deletion analogues, including polypeptide fragments, lack one or more residues of the native protein that are not essential for function or immunogenic activity. Insertion analogs involve adding, for example, an amino acid(s) to a non-terminal point of a polypeptide. This analogue may include, for example, but is not limited to, the insertion of an immunoreactive epitope or simply a single residue. Addition analogues, including polypeptide fragments, involve the addition of one or more amino acids at one or both ends of a protein and include, for example, fusion proteins. Combinations of the above analogues are also considered.

Substitute analogs typically replace one wild-type amino acid with another at one or more sites within a protein and can be designed to modulate one or more properties of a polypeptide without completely losing other functions or properties. In one aspect, the substitutions are conservative substitutions. A conservative amino acid substitution is a replacement of an amino acid with an amino acid having a side chain or similar chemical character. Such amino acids for making conservative substitutions include amino acids with an acidic side chain (glutamic acid, aspartic acid); main side chain (arginine, lysine, histidine); polar amide side chain (glutamine, asparagine); hydrophobic aliphatic side chain (leucine, isoleucine, valine, alanine, glycine); aromatic side chain (phenylalanine, tryptophan, tyrosine); small side chain (glycine, alanine, serine, threonine, methionine); or an aliphatic hydroxyl side chain (serine, threonine).

In one aspect, the analogs are substantially homologous or substantially identical to the recombinant VWF from which they are derived. Analogs include those that retain at least a portion of the biological activity of the wild-type polypeptide, for example, blood clotting activity.

Contemplated polypeptide variants include, but are not limited to, polypeptides chemically modified by methods such as ubiquitination, glycosylation including polysialylation (or polysialylation), conjugation with therapeutic or diagnostic agents, labeling, covalent attachment of a polymer such as PEGylation (polyethylene glycol derivatization), introduction of non-hydrolyzable bonds, and insertion or substitution by chemical synthesis of amino acids such as ornithine, which are not normally found in human proteins. The variants retain the same or substantially the same binding properties of the unmodified molecules of the invention. Such chemical modification may involve direct or indirect (eg, via a linker) attachment of the agent to the VWF polypeptide. In the case of indirect attachment, it is contemplated that the linker may be hydrolyzable or non-hydrolysable.

Preparation of PEGylated polypeptide analogs will, in one aspect, comprise the steps of (a) reacting the polypeptide with a polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions under which the polypeptide of the linking construct is attached to one or more PEG groups, and (b) obtaining a product (s) reactions. In general, optimal reaction conditions for acylation reactions are determined based on known parameters and the desired outcome. For example, the higher the PEG:protein ratio, the higher the percentage of polyPEgylated product. In some embodiments, the binding construct has a single PEG moiety at the N-terminus. Polyethylene glycol (PEG) can be attached to a clotting factor, for example, to provide a longer half-life in vivo. The PEG group may have any suitable molecular weight and be linear or branched. The average molecular weight of PEG ranges from about 2 kDa (kDa) to about 100 kDa, from about 5 kDa to about 50 kDa, or from about 5 kDa to about 10 kDa. In some aspects, PEG groups are attached to the coagulation factor by acylation or reductive alkylation via a natural or engineered reactive group on the PEG moiety (e.g., an aldehyde, amino, thiol, or ester group) to a coagulation factor reactive group (e.g., aldehyde, amino- or ester group) or any other method known in the art.

Methods for producing a polysialylated polypeptide are described in US 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 NaIO ₄ is stirred in the dark at room temperature to oxidize the CA. The activated SA 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 for 18 hours at room temperature in the dark with gentle shaking. Free reactants are optionally separated from the rVWF-polysialic acid conjugate, for example, by 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).

Moreover, in another aspect, it is contemplated that the polypeptide of the invention is a fusion protein with a second agent that is a polypeptide. In one embodiment, the second agent, which is a polypeptide, is, without limitation, pher

- 33 045553 ment, growth factor, antibody, cytokine, chemokine, cell surface receptor, cell surface receptor extracellular domain, cell adhesion, molecule, or fragment, or active domain of a protein described above. 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. The subject fusion protein is produced by chemical or recombinant methods well known in the art. In some embodiments, the fusion protein is an rVWF-FVIII fusion protein. In some embodiments, the fusion protein is an rVWF-FVIII fusion protein, wherein active FVIII is inserted into a VWF motif. In some embodiments, the fusion protein is an rVWF-FVIII fusion protein, where the active FVIII is inserted into the VWF motif such that the VWF is full-length. In some embodiments, the fusion protein is an rVWFFVIII fusion protein, wherein active FVIII is inserted into a VWF motif, with portions of the VWF sequence removed and replaced with FVIII sequence. In some embodiments of the rVWF-FVIII fusion protein, the FVIII is FVIII with the B domain removed. In some embodiments of the rVWF-FVIII fusion protein, the N-glycosylation-rich domain replaces the FVIII-B domain. In some embodiments of the rVWF-FVIII fusion protein, the vWF-N glycosylation-rich domain is fused to full-length FVIII and/or truncated forms thereof.

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, an 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 , the FVIII peptide containing positions 1333 to 2351 of the FVIII light chain peptide, and the VWF peptide containing positions 2620 to 2813 of the VWF peptide.

In this embodiment of the rVWF-FVIII fusion protein, the amino acid position is counted from the first position including Pro and/or signal peptide. In this embodiment of the rVWF-FVIII fusion protein, position 764 in VWF corresponds to position 1 of the 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 FIG. 64.

In some embodiments of the rVWF-FVIII fusion protein, the fusion protein comprises: an FVIII peptide comprising FVIII heavy chain positions 19 to 760 of the FVIII heavy chain peptide, a VWF peptide comprising positions 2218 to 2593 of the VWF peptide, and an FVIII peptide comprising positions 1333 2351 each of FVIII light chain peptides.

In this embodiment of the rVWF-FVIII fusion protein, the amino acid position is counted from the first position including Pro and/or signal peptide. In this embodiment of the rVWF-FVIII fusion protein, position 764 in VWF corresponds to position 1 of the 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 FIG. 65.

In another aspect, it is also contemplated that the prepro-VWF and pro-VWF polypeptides will provide a therapeutic effect in the compositions of the present invention. For example, US Pat. No. 7,005,502 describes a pharmaceutical preparation containing significant amounts of pro-VWF that induces thrombin formation in vitro. In addition to recombinant, biologically active fragments, variants or other analogs of naturally occurring mature VWF, the present invention contemplates the use of recombinant biologically active fragments, variants or analogs of prepro-VWF (set forth in SEQ ID NO: 2) or pro-VWF polypeptides (amino acid residues from 23 to 764 SEQ ID NO: 2) in the compositions described herein.

Polynucleotides encoding fragments, variants and analogs can be readily prepared by one skilled in the art of encoding biologically active fragments, variants or analogs of a naturally occurring molecule that have the same or similar biological activity as the naturally occurring molecule. In various aspects, these polynucleotides are produced using PCR techniques, enzymatic digestion/ligation of a DNA encoding molecule, and the like. Thus, one skilled in the art will be able to create single base changes in a DNA strand, resulting in an altered codon and a missense mutation, using any method known in the art, including, but not limited to, site-directed mutagenesis. As used herein, the phrase moderately stringent hybridization conditions means, for example, hybridization at 42°C in 50% formamide and washing at 60°C in 0.1 x SSC, 0.1% SDS. Those skilled in the art will appreciate that these conditions vary depending on the length and nucleobase content of the GC sequences to be hybridized. Formulas standard in the art are suitable for determining the exact hybridization conditions. See Sambrook et al., 9.47-9.51 in Molecular Cloning, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

Virus inactivation

In some embodiments, the method described herein further includes a viral inactivation step. The virus inactivation step can occur before, after or

- 34 045553 simultaneously with the washing step and/or the elution step, but before the collection step. Virus inactivation treatments can inactivate lipid enveloped viruses. In some embodiments, the virus inactivation treatment is a solvent and detergent (S/D) treatment. In some embodiments, the virus inactivation treatment includes the use of ethylene glycol, propylene glycol and/or one or more organic solvents.

As used herein, the term virus inactivation or virus inactivation refers to the process by which the virus can no longer infect cells, replicate or multiply, and the removal of the virus per se. As such, the term virus inactivation generally refers to the process of obtaining the liquid described herein completely free of infectious viral contaminants. Any degree of viral inactivation is desirable using the methods described herein. However, it is desirable to achieve the degree of viral inactivation necessary to comply with stringent safety regulations for pharmaceuticals. These guidelines are formulated by WHO and are well known to those skilled in the art.

The methods disclosed herein may further include the step of removing the virus from the mixture after incubation. As used herein, the term virus removal or virus elimination refers to a process that destroys the virus in a mixture disclosed herein so that viral particles are effectively removed from the mixture. The virus may be a viable or inactivated virus. Removal is typically accomplished by size exclusion chromatography or positive adsorption chromatography where the protein of interest is coupled to a chromatography resin, including, for example, an anion exchange resin or a cation exchange resin, as described herein. Once removed, the amount of virus remaining is an amount that substantially does not cause long-term or persistent harmful effects when administered to a subject in need thereof, including, for example, a human.

In one embodiment, the mixture contains substantially no virus after removal of the virus. As used herein, the term substantially free of virus means that only trace amounts of virus can be detected or confirmed by an instrument or process used to detect or confirm the presence or activity of a virus, and that such trace amounts of virus are not sufficient to be harmful to human health. In one aspect of this embodiment, the mixture is completely free of virus after removal of the virus. As used herein, the term completely virus-free means that the presence of the virus cannot be detected or confirmed within the detection range of the instrument or process used to detect or confirm the presence or activity of the virus. The protein contained in the mixture, which is substantially free or completely free of virus, can be used to make a pharmaceutical composition that is safe for administration to humans because the virus is not sufficient to cause harm to human health.

In other aspects of this embodiment, the mixture after removal of the virus contains less than 10 PFU/ml of virus, for example, less than 1 PFU/ml of virus, less than 1x10'1 PFU/ml of virus, 1x10'2 ^PFU /ml of virus, or 1x10'3 PFU/ml virus.

In other aspects of this embodiment, the mixture, after removal of the virus, contains less than the ID50 of the virus, e.g., at least 10 times less than the ID50 of the virus, at least 100 times less than the ID50 of the virus, at least 200 times less than the ID50 for a virus, at least 300 times less than the ID50 for a virus, at least 400 times less than the ID50 for a virus, at least 500 times less than the ID50 for a virus, at least 600 times less than the ID50 of a virus, at least 700 times less than the ID50 of a virus, at least 800 times less than the ID50 of a virus, at least 900 times less than the ID50 of a virus, or at least at least 1000 times less than the ID50 for the virus.

Virus inactivation can be performed simultaneously with or without protein purification. In some embodiments, the method includes immobilizing a protein on a support and treating the immobilized protein with a detergent-solvent mixture containing a nonionic detergent and an organic solvent. In some embodiments, the carrier is a chromatography resin. In some embodiments, the detergent-solvent mixture contains 1% Triton X-100, 0.3% tri-K-butyl phosphate, and 0.3% polysorbate 80 (Tween 80). Treatment with the solvent-detergent mixture can be continued for a long time, for example 30 minutes to 1 hour, while the protein remains immobilized on the chromatography resin, for example, a cation exchange resin; and/or solvent-detergent treatment may occur at a temperature of from 2 to 10°C. This approach to inactivating viruses can unexpectedly reduce the formation of protein aggregates during detergent-solvent treatment by a significant amount, for example, more than 50%, compared to solvent-detergent treatment when the protein is not immobilized in solution.

In some embodiments, a method of inactivating a lipid envelope virus includes the steps of: i) providing a liquid containing a protein having the activity; ii) mixing the organic solvent and surfactant with the liquid, 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 not exceeding about 20°C; where the mixture after incubation is practically free of virus containing a viable lipid envelope; and wherein the protein after incubation has an activity that is at least 25% of the activity provided in step (i).

In other embodiments, a protein substantially free of lipid envelope-containing virus may be produced by a process comprising the following steps: i) providing a liquid containing the protein having the activity; ii) mixing the organic solvent and surfactant with the liquid, 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 not exceeding about 20°C; where the mixture after incubation is practically free of virus containing a viable lipid envelope; and wherein the protein, after incubation, has an activity that is at least 25% of the activity predicted in step (a).

In another embodiment, a method of inactivating a lipid envelope virus includes the steps of: i) providing a liquid containing a blood clotting protein having activity (eg, VWF); ii) mixing the organic solvent and surfactant with the liquid, 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 not exceeding about 20°C; where the mixture after incubation is practically free of virus containing a viable lipid envelope; and wherein factor VIII, after incubation, has an activity that is at least 25% of the activity provided in step (i).

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

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-(tert-butyl)phosphate, di-(n-hexyl)phosphate, di-(2ethylhexyl)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-(tert-butyl)phosphate, tri-(nhexyl)phosphate, tri-(2-ethylhexyl)phosphate, and/or tri-(n-decyl) phosphate.

In some cases, the final concentration of organic solvent is from about 0.1% (v/v) to about 5.0% (v/v), from 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), about 0.3% (v/v).

In some cases, the surfactant is selected from an ionic surfactant, a zwitterionic surfactant, and/or a nonionic surfactant. The ionic surfactant may be an anionic surfactant or a cationic surfactant.

In some embodiments, the anionic surfactant is selected from alkyl sulfate, alkyl ether sulfate, docusate, fluorosulfonate surfactant, alkyl benzene sulfonate, alkyl aryl phosphate, alkyl ether phosphate; alkyl carboxylate, sodium lauroyl sarcosinate and/or a carboxylate fluorosurfactant. 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 sodium dioctyl sulfosuccinate.

In some embodiments, the sulfonate fluorinated surfactant is selected from perfluorooctane sulfonate (PFOS) and/or perfluorobutane sulfonate. In some embodiments, the alkyl carboxylate is selected from a fatty acid salt and/or sodium stearate. In some embodiments, the carboxylate fluorosurfactant is perfluoronanoate and perylfluorooctanoate. In some embodiments, the cationic surfactant is selected from alkyltrimethylammonium salt, cetylpyridinium chloride (CPC), polyethoxylated tal amine (POEA), benzalkonium chloride (BAC), benzethonium chloride (BZT), 5-bromo-5-nitro-1,3-dioxane, dimethyldioctadecylammonium chloride, dioctadecyldimethylammonium bromide (DODAB), pH-dependent primary amine, pH-dependent secondary amine and/or pH-dependent tertiary amine. In some embodiments, the alkyltrimethylammonium salt is selected from cetyltrimethylammonium bromide (CTAB) and/or cetyltrimethylammonium chloride (CTAC). In some embodiments, the primary amine becomes positively charged at pH <10 or the secondary amine becomes charged at pH <4.

In some embodiments, the cationic surfactant is octenidine dihydrochloride.

In some embodiments, the zwitterionic surfactant is selected

- 36 045553 from 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), sultaine, betaine and/or lecithin. In some embodiments, the sultaine is cocamidopropylhydroxysultaine. In some embodiments, the betaine is cocamidopropyl betaine.

In some embodiments, the nonionic surfactant is selected from polyoxyethylene glycol sorbitan alkyl ether, poloxamer, alkylphenol polyglycol ether, polyethylene glycol alkyl aryl ether, polyoxyethylene glycol alkyl ether, 2-dodecoxyethanol (LUBROL®-PX), polyoxyethylene glycol octylphenol ether, just th alkylphenol ester polyoxyethylene glycol, phenoxypolyethoxylethanol, glucoside alkyl ether, maltoside alkyl ether, thioglucoside alkyl ether, digitonin, glycerol alkyl ester, alkylaryl polyether sulfate, alcohol sulfonate, sorbitan alkyl ether, ethanolamine cocamide, 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 6 5 sorbitan monooleate (TWEEN® 65), polysorbate 80 sorbitan monooleate (TWEEN® 80) and/or polysorbate 81 sorbitan monooleate (TWEEN® 81).

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).

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.

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

In some embodiments, the phenoxypolyethoxylethanol is selected from nonylphenoxypolyethoxylethanol and/or octylphenoxypolyethoxylethanol.

In some embodiments, the alkyl ester of the glucoside is an octyl glucopyranoside. In some embodiments, the maltoside alkyl ester is dodecyl maltopyranoside. In some embodiments, the alkyl ester of the thioglucoside is heptylthioglucopyranoside. In some embodiments, the glycerol alkyl ester is glycerol laurate. In some embodiments, the cocamide ethanolamine is selected from cocamide monoethanolamine and/or cocamide ethanolamine.

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

Useful methods for inactivating viruses are described, for example, in US Pat. Nos. 6,190,609 and 9,315,560 and US Application Publication No. 2017/0327559, the contents of which are incorporated herein by reference in their entirety.

Virus inactivation can be carried out as is known to those skilled in the art. For example, tri(n-butyl) phosphate (TNBP) solvent and detergents such as, but not limited to, Polysorbate 80 and Triton X-100 are effective in inactivating lipid-enveloped viruses. Inactivation of viruses can be carried out at room temperature, for example from 14°C to about 25°C, for about 1 hour or more. In some cases, the incubation time does not exceed two hours.

In some embodiments, the virus inactivation treatment is terminated by adding a buffer containing sodium citrate to the virus-inactivated material. In some cases, the sodium citrate buffer contains from about 40 to about 100 mM sodium citrate buffer, such as about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM citrate buffer sodium

VWF maturation

Furin is part of a family of proteins called SPC (subtilisin-like proprotein convertases), PC (proprotein convertases) or in some cases PACE (paired 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. Within the scope of this invention, methods provide methods for maturing pro-VWF (pro-rVWF) into a mat-VWF/VWF-PP complex (mat-rVWF/rVWF-PP) by treatment with furin. Any of these furin family members can be used in VWF maturation methods.

In some embodiments, pro-VWF is matured using furin, an anion exchange column or resin, a cation exchange column or resin, or as part of a size separation chromatography method. 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, matured on a cation exchange column or resin, and/or as part of a cation exchange chromatography procedure. In some embodiments, pro-VWF is matured with furin as part of a size exclusion chromatography procedure. Such methods have been described, for example, in US Pat. No. 8,058,411, which is incorporated herein by reference in its entirety for all purposes.

To facilitate the maturation process and provide pro-VWF immobilized on the resin at an increased concentration, in some embodiments the chromatography resin is placed in a chromatography column. Since the concentration of pro-VWF during its in vitro maturation affects the maturation efficiency, it is advantageous to place the chromatography resin on the column. In addition, the use of chromatographic columns allows for better control of maturation parameters in a more reproducible manner and makes it easier to perform in vitro maturation of VWF. In some embodiments, the furin concentration is about 1, about 2, about 3, or about 4 units of recombinant active furin per VWF:Ag ME (10 μg pro-rVWF). In some embodiments, the furin concentration is about 2-3 units of recombinant active furin per VWF:Ag ME (10 μg pro-rVWF). In some embodiments, the furin concentration is about 1-2 units of recombinant active furin per VWF:Ag ME (10 μg prorVWF). In some embodiments, the furin concentration is about 2 units of recombinant active furin per VWF:Ag ME (10 μg pro-rVWF).

In some embodiments, when pro-VWF is immobilized on an anion exchange resin and incubated with a solution exhibiting pro-VWF convertase activity, the conductivity measured at 25°C is below 25 mS/cm. In some embodiments, when pro-VWF is immobilized on an anion exchange resin and incubated with a solution exhibiting pro-VWF convertase activity, the conductivity measured at 25°C is below 20 mS/cm. In some embodiments, when proVWF is immobilized on an anion exchange resin and incubated with a solution exhibiting pro-VWF convertase activity, the conductivity measured at 25°C is below 16 mS/cm. In some embodiments, when pro-VWF is immobilized on an anion exchange resin and incubated with a solution exhibiting pro-VWF convertase activity, the conductivity measured at 25°C is in the range of 16 to 25 mS/cm. In some embodiments, when pro-VWF is immobilized on an anion exchange resin and incubated with a solution exhibiting pro-VWF convertase activity, the conductivity measured at 25°C is in the range of 20 to 25 mS/cm. Pro-rVWF as well as mat-rVWF can be effectively immobilized on anion exchange resins at these conductivity levels. Therefore, the buffers used during this method must be suitably adapted to maintain conductivity levels. In some embodiments, the conductivity is such that the furin and/or PACE enzyme is in an active form and wholly or partially in the mobile phase.

In some embodiments, mat-rVWF elutes from the anion exchange resin with a conductivity measured at 25°C of at least 40 mS/cm. In some embodiments, mat-rVWF elutes from the anion exchange resin with a conductivity measured at 25°C of at least 60 mS/cm. In some embodiments, mat-rVWF elutes from the anion exchange resin with a conductivity measured at 25°C of at least 80 mS/cm. In some embodiments, mat-rVWF elutes from the anion exchange resin with a conductivity measured at 25°C ranging from 40 mS/cm to 80 mS/cm. In some embodiments, mat-rVWF elutes from the anion exchange resin with a conductivity measured at 25°C ranging from 60 mS/cm to 80 mS/cm. In some embodiments, the desired rVWF particles begin to elute at a conductivity of about 12 to 16 mS/cm/25°C using an anion exchange resin (eg, TMAE). In some embodiments, the majority (bulk) of the desired rVWF particles were eluted with the anion exchange resin in the range of about 55 to 60 mS/cm/25°C. In some embodiments, the desired rVWF particles begin to elute at a conductivity of about 18 to 24 mS/cm/25°C using the cation exchange resin. In some embodiments, the majority (bulk) of the desired rVWF particles were eluted with the cation exchange resin in the range of about 36 to 42 mS/cm/25°C. In some embodiments, the desired rVWF is a mature rVWF (eg, mat-rVWF). In some embodiments, the main amount (bulk) includes at least about 30, 40, 50, 60, 70, 80, 90% or more of the total amount of desired substances eluted.

In some embodiments, additional washing steps are used before eluting the mat-rVWF from the anion exchange resin. In some embodiments, additional washing steps are used before eluting the mat-rVWF cation exchange resin.

For their proteolytic activity, many proteases require cofactors such as ions

- 38 045553 divalent metals. Furin and members of the furin family of proteins require calcium ions for activity. Therefore, if furin is used for in vitro maturation of pro-rVWF, calcium salts are used. In some embodiments, the calcium salt is a soluble calcium salt. In some embodiments, the calcium salt is calcium chloride (CaCl2). In some embodiments, the calcium salt is calcium acetate. In some embodiments, other divalent metal ions are used, including, for example, without limitation, Be ²⁺ , Ba ²⁺ , Mg ²⁺ , Mn ²⁺ , Sr ²⁺ , Zn ²⁺ , Co ²⁺ , Ni ²⁺ , Cd ²⁺ and/or Cu ²⁺ . In some embodiments, a combination of two or more divalent cations is used. In some embodiments, Ca ²⁺ and Mg ²⁺ are used in combination. In some embodiments, the magnesium salt is a soluble magnesium salt. In some embodiments, the magnesium salt is magnesium chloride (MgCl2). In some embodiments, the furin protein family composition for use in ripening contains a soluble calcium salt at a concentration of 0.01 to 10 mM. In some embodiments, the furin protein family formulation for use in ripening contains a soluble magnesium salt at a concentration of 0.01 to 10 mM. In some embodiments, the furin protein family formulation for use in ripening contains CaCl ₂ at a concentration of 0.01 to 10 mM. In some embodiments, the furin protein family composition for use in ripening contains MgCl ₂ at a concentration of 0.01 to 10 mM. In some embodiments, the furin protein family formulation for use in ripening contains CaCl ₂ at a concentration of 0.1 to 5 mM. In some embodiments, the furin protein family composition for use in ripening contains MgCl ₂ at a concentration of 0.1 to 5 mM. In some embodiments, the furin protein family formulation for use in ripening contains CaCl ₂ at a concentration of 0.2 to 2 mM. In some embodiments, the furin protein family formulation for use in ripening contains MgCl ₂ at a concentration of 0.2 to 2 mM. In some embodiments, the furin family protein composition for use in ripening comprises furin. In some embodiments, the furin concentration is about 1, about 2, about 3, or about 4 units of recombinant active furin per VWF:Ag ME (10 μg pro-rVWF). In some embodiments, the furin concentration is about 2-3 units of recombinant active furin per VWF:Ag ME (10 μg pro-rVWF). In some embodiments, the furin concentration is about 1-2 units of recombinant active furin per VWF:Ag ME (10 μg pro-rVWF). In some embodiments, the furin concentration is about 2 units of recombinant active furin per VWF:Ag ME (10 μg pro-rVWF).

The incubation time of furin with immobilized pro-rVWF may vary depending on the system used. The efficiency of the ripening process is also influenced by factors such as temperature, buffering, etc. Typically, the ripening process is completed in less than 48 hours. In some embodiments, the ripening process can occur in less than 1 minute. In some embodiments, the ripening process may occur in less than 40, 36, 30, 24, 20, 16, 10, 5, 2, 1 hour or less. In some embodiments, the pro-rVWF maturation incubation is carried out for less than 1 minute to 48 hours. In some embodiments, the pro-rVWF maturation incubation is carried out for from 10 minutes to 42 hours. In some embodiments, the pro-rVWF maturation incubation is carried out for from 10 minutes to 42 hours. rVWF is carried out for 20 minutes to 36 hours. In some embodiments, pro-rVWF maturation incubation is carried out for 30 minutes to 24 hours. In some embodiments, due to the high specificity of furin, VWF overactivation (further proteolytic degradation) does not occur even after long incubation time.

In some embodiments, the ripening process also depends on the temperature selected during incubation. The optimal enzymatic activity of furin depends on temperature.

In some embodiments, pro-rVWF maturation incubation is carried out at a temperature of 2 to 40°C. In some embodiments, pro-rVWF maturation incubation is carried out at a temperature of 4 to 37°C. In some embodiments, prorVWF maturation incubation is performed at low temperatures, such as 2°C. In some embodiments, the maximum temperature used is below 50°C to avoid and/or prevent protein degradation. In some embodiments, the maximum temperature used is below 45°C to avoid and/or prevent protein degradation.

In some embodiments, pro-VWF (or pro-rVWF) is converted to mat-VWF (or matrVWF) by treatment with furin or a member of the furin family as described above. In some embodiments, treatment with furin results in at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% conversion of prorVWF to mat- rVWF and rVWF-PP. In some embodiments, after sizing in the presence of the addition of at least one chelating agent and/or increasing the pH to a pH of at least 7, less than 5% rVWF-PP, less than 4% rVWF-PP, less than 3% rVWF-PP remains , less than 2% rVWFPP, 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 in the eluate.

- 39 045553

table 2

Estimated removal of pro-VWF (based on furin treatment)

Stage Loading, VWF-PP admixture Eluate, VWF-PP admixture % (mass/mass) % (mass/mass) AEX -70% -0.5% SEX -0.5% -0.5% SEC -0.5% -0.5%

VWF multimers

Estimation of the amount and percentage of rVWF multimers can be made using methods known in the art, including, without limitation, methods using electrophoresis and size exclusion chromatography methods for separating VWF multimers by size, for example, as discussed in Cumming et al., (J Clin Pathol., 1993 May; 46(5): 470-473, which is incorporated herein by reference for all purposes and in particular for all recommendations relating to the evaluation of VWF multimers). Such methods may further include immunoblotting techniques (such as Western blotting) in which the gel is immunoblotted with a radiolabeled anti-VWF antibody followed by chemiluminescent detection (see, for example, Wen et al., J. Clin. Lab. Anal. , 1993, 7: 317-323, which is incorporated herein by reference in its entirety for all purposes and in particular as it relates to the evaluation of VWF multimers). Additional tests for VWF include VWF:antigen (VWF:Ag), VWF:ristocetin cofactor (VWF:RCof), and VWF:collagen binding activity assay (VWF:CBA), which are often used to diagnose and classify von Willebrand disease (see for example, Favaloro et al., Pathology, 1997, 29(4): 341-456; Sadler, JE, Annu Rev Biochem, 1998, 67:395-424 and Turecek et al., Semin Thromb Hemost, 2010,

36:510-521, which are hereby incorporated by reference in their entirety for all purposes and, in particular, for all procedures related to VWF assays). In some embodiments, the matrVWF obtained using these methods includes any multidimensional pattern present in the rVWF boot sample. In some embodiments, the mat-rVWF produced using these methods includes physiologically occurring multimer patterns as well as ultra-large VWF multimer patterns.

VWF analyzes

In primary hemostasis, VWF serves as a bridge between platelets and certain extracellular matrix components 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: 510521).

The VWF:ristocetin cofactor (VWF:RCof) assay is based on agglutination of fresh or formalin-fixed platelets induced by the antibiotic ristocetin in the presence of VWF. The degree of platelet agglutination depends on the concentration of VWF and can be measured by a turbidimetric method, for example, using an aggregometer (Weiss et al., J. Clin. Invest., 1973, 52: 2708-2716; Macfarlane et al., Thromb. Diath. Haemorrhage ., 1975, 34: 306-308). As provided herein, the specific activity of the VWF ristocetin cofactor (VWF:RCo) of the present invention is typically described in units of mU/μg VWF, as measured using in vitro assays.

In some embodiments, mat-rVWF purified by the methods of this 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/mcg. In some embodiments, mat-rVWF used in the methods described herein has a specific activity of 20 to 150 mU/μg. In some embodiments, mat-rVWF has a specific activity of 30 to 120 mU/μg. In some embodiments, mat-rVWF has a specific activity of 40 to 90 mU/μg. In some embodiments, mat-rVWF has a specific activity selected from options 1 to 133 listed in table. 3 below.

- 40 045553

Table 3

Exemplary embodiments of the specific rVWF activity found in the compositions and methods provided herein

(mU/mcg) (mU/mcg) (mU/mcg) (mU/mcg) 20 Var. 1 BY Var. 35 40-150 Var. 68 70-120 Var. 101 22.5 Var. 2 115 Var. 36 40-140 Var. 69 70-110 Var. 102 25 Var. 3 120 Var. 37 40-130 Var. 70 70-100 Var. 103 27.5 Var. 4 125 Var. 38 40-120 Var. 71 70-90 Var. 104 thirty Var. 5 130 Var. 39 40-110 Var. 72 70-80 Var. 105 32.5 Var. 6 135 Var. 40 40-100 Var. 73 80-150 Var. 106 35 Var. 7 140 Var. 41 40-90 Var. 74 80-140 Var. 107 37.5 Var. 8 145 Var. 42 40-80 Var. 75 80-130 Var. 108 40 Var. 9 150 Var. 43 40-70 Var. 76 80-120 Var. 109 42.5 Var. 10 20-150 Var. 44 40-60 Var. 77 80-110 Var. BY 45 Var. AND 20-140 Var. 45 40-50 Var. 78 80-100 Var. 111 47.5 Var. 12 20-130 Var. 46 50-150 Var. 79 80-90 Var. 112 50 Var. 13 20-120 Var. 47 50-140 Var. 80 90-150 Var. 113 52.5 Var. 14 20-110 Var. 48 50-130 Var. 81 90-140 Var. 114 55 Var. 15 20-100 Var. 49 50-120 Var. 82 90-130 Var. 115 57.5 Var. 16 20-90 Var. 50 50-110 Var. 83 90-120 Var. 116 60 Var. 17 20-80 Var. 51 50-100 Var. 84 90-110 Var. 117 62.5 Var. 18 20-70 Var. 52 50-90 Var. 85 90-100 Var. 118 65 Var. 19 20-60 Var. 53 50-80 Var. 86 100-150 Var. 119 67.5 Var. 20 20-50 Var. 54 50-70 Var. 87 100-140 Var. 120 70 Var. 21 20-40 Var. 55 50-60 Var. 88 100-130 Var. 121 72.5 Var. 22 30-150 Var. 56 60-150 Var. 89 100-120 Var. 122 75 Var. 23 30-140 Var. 57 60-140 Var. 90 100-110 Var. 123 77.5 Var. 24 30-130 Var. 58 60-130 Var. 91 110-150 Var. 124 80 Var. 25 30-120 Var. 59 60-120 Var. 92 110-140 Var. 125 82.5 Var. 26 30-110 Var. 60 60-110 Var. 93 110-130 Var. 126 85 Var. 27 30-100 Var. 61 60-100 Var. 94 110-120 Var. 127 87.5 Var. 28 30-90 Var. 62 60-90 Var. 95 120-150 Var. 128 90 Var. 29 30-80 Var. 63 60-80 Var. 96 120-140 Var. 129 92.5 Var. thirty 30-70 Var. 64 60-70 Var. 97 120-130 Var. 130 95 Var. 31 30-60 Var. 65 70-150 Var. 98 130-150 Var. 131 97.5 Var. 32 30-50 Var. 66 70-140 Var. 99 130-140 Var. 132 100 Var. 33 30-40 Var. 67 70-130 Var. 100 140-150 Var. 133 105 Var. 34

Var. = Option.

The Mat-rVWF of the present invention is highly multimeric, containing from about 10 to about 40 subunits. In additional embodiments, the multimeric rVWF produced using the methods of this invention contains about 10-30, 12-28, 14-26, 16-24, 18-22, 20-21 subunits. In some embodiments, rVWF is present in multimers that range in size from dimers to multimers containing more than 40 subunits (>10 million daltons). The largest multimers provide multiple binding sites that can interact with both platelet receptors and sites of subendothelial matrix damage and are the most hemostatically active form of VWF. In some embodiments, the mat-rVWF of this invention contains ultra-large multimers (ULMs,

-41 045553 English ultralarge multimers). In general, large and extra-large multimers are the most hemostatically effective (see, for example, Turecek, P., Hamostaseologie, (Vol. 37): Supplement 1, pages S15-S25 (2017)). In some embodiments, mat-rVWF has from 500 to 20,000 kDa. In some embodiments, any desired pattern of multimers can be obtained using the methods described. In some embodiments, when anion exchange and/or cation exchange methods are used, the pH, conductivity, and/or counterion concentration of the buffers in one or more wash step(s) or gradient buffers can be varied to obtain the desired multimer pattern. In some embodiments, size exclusion chromatography techniques are then used, collection criteria can be used to obtain the desired multimer pattern. In some embodiments, the described multimer pattern comprises ultra-large multimers. In some embodiments, the 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, at least 20,000 kDa. In some embodiments, the ultra-large multimers are from about 10,000 to 20,000 kDa. In some embodiments, the ultra-large multimers are between about 11,000 and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 12,000 and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 13,000 and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 14,000 and 20,000 kDa. In some embodiments, the ultra-large multimers are from about 15,000 to 20,000 kDa. In some embodiments, the ultra-large multimers are between about 16,000 and 20,000 kDa. In some embodiments, the ultra-large multimers are between about 17,000 and 20,000 kDa. In some embodiments, the ultra-large multimers are from about 18,000 to 20,000 kDa. In some embodiments, the ultra-large multimers are between about 19,000 and 20,000 kDa. In some embodiments, the mat-rVWF obtained using these methods includes any multidimensional pattern present in the rVWF boot sample. In some embodiments, the mat-rVWF produced using these methods includes physiologically occurring multimer patterns as well as ultra-large VWF multimer patterns.

In some embodiments, the mat-rVWF composition prepared by the purification method described herein has a distribution of rVWF oligomers such that 95% of the oligomers contain from 6 to 20 subunits. In some embodiments, the matrVWF composition has a rVWF oligomer distribution characterized in that 95% of the oligomers have a subunit range selected from options 458 to 641 shown in Table. 4.

Table 4 Example distributions of rVWF oligomers found in the compositions and methods used herein

Subunits Subunits Subunits Subunits 2-40 Var. 458 6-16 Var. 504 12-20 Var. 550 20-28 Var. 596

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2-38 Bap. 459 6-14 Bap. 505 12-18 Bap. 551 20-26 Bap. 597 Bap. Bap. Bap. Bap. 2-36 460 6-12 506 12-16 552 20-24 598 Bap. Bap. Bap. Bap. 2-34 461 6-10 507 12-14 553 20-22 599 Bap. Bap. Bap. Bap. 2-32 462 6-8 508 14-40 554 22-40 600 Bap. Bap. Bap. Bap. 2-30 463 8-40 509 14-38 555 22-38 601 Bap. Bap. Bap. Bap. 2-28 464 8-38 510 14-36 556 22-36 602 Bap. Bap. Bap. Bap. 2-26 465 8-36 511 14-34 557 22-34 603 Bap. Bap. Bap. Bap. 2-24 466 8-34 512 14-32 558 22-32 604 Bap. Bap. Bap. Bap. 2-22 467 8-32 513 14-30 559 22-30 605 Bap. Bap. Bap. Bap. 2-20 468 8-30 514 14-28 560 22-28 606 Bap. Bap. Bap. Bap. 2-18 469 8-28 515 14-26 561 22-26 607 Bap. Bap. Bap. Bap. 2-16 470 8-26 516 14-24 562 22-24 608 Bap. Bap. Bap. Bap. 2-14 471 8-24 517 14-22 563 24-40 609 Bap. Bap. Bap. Bap. 2-12 472 8-22 518 14-20 564 24-38 610 Bap. Bap. Bap. Bap. 2-10 473 8-20 519 14-18 565 24-36 611 Bap. Bap. Bap. Bap. 2-8 474 8-18 520 14-16 566 24-34 612 Bap. Bap. Bap. Bap. 4-40 475 8-16 521 16-40 567 24-32 613

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4-38 Bap. 476 8-14 Bap. 522 16-38 Bap. 568 24-30 Bap. 614 4-36 Bap. 477 8-12 Bap. 523 16-36 Bap. 569 24-28 Bap. 615 4-34 Bap. 478 8-10 Bap. 524 16-34 Bap. 570 24-26 Bap. 616 4-32 Bap. 479 10-40 Bap. 525 16-32 Bap. 571 26-40 Bap. 617 4-30 Bap. 480 10-38 Bap. 526 16-30 Bap. 572 26-38 Bap. 618 4-28 Bap. 481 10-36 Bap. 527 16-28 Bap. 573 26-36 Bap. 619 4-26 Bap. 482 10-34 Bap. 528 16-26 Bap. 574 26-34 Bap. 620 4-24 Bap. 483 10-32 Bap. 529 16-24 Bap. 575 26-32 Bap. 621 4-22 Bap. 484 10-30 Bap. 530 16-22 Bap. 576 26-30 Bap. 622 4-20 Bap. 485 10-28 Bap. 531 16-20 Bap. 577 26-28 Bap. 623 4-18 Bap. 486 10-26 Bap. 532 16-18 Bap. 578 28-40 Bap. 624 4-16 Bap. 487 10-24 Bap. 533 18-40 Bap. 579 28-38 Bap. 625 4-14 Bap. 488 10-22 Bap. 534 18-38 Bap. 580 28-36 Bap. 626 4-12 Bap. 489 10-20 Bap. 535 18-36 Bap. 581 28-34 Bap. 627 4-10 Bap. 490 10-18 Bap. 536 18-34 Bap. 582 28-32 Bap. 628 4-8 Bap. 491 10-16 Bap. 537 18-32 Bap. 583 28-30 Bap. 629 6-40 Bap. 492 10-14 Bap. 538 18-30 Bap. 584 30-40 Bap. 630

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6-38 Var. 493 10-12 Var. 539 18-28 Var. 585 30-38 Var. 631 6-36 Var. 494 12-40 Var. 540 18-26 Var. 586 30-36 Var. 632 6-34 Var. 495 12-38 Var. 541 18-24 Var. 587 30-34 Var. 633 6-32 Var. 496 12-36 Var. 542 18-22 Var. 588 30-32 Var. 634 6-30 Var. 497 12-34 Var. 543 18-20 Var. 589 32-40 Var. 635 6-28 Var. 498 12-32 Var. 544 20-40 Var. 590 32-38 Var. 636 6-26 Var. 499 12-30 Var. 545 20-38 Var. 591 32-36 Var. 637 6-24 Var. 500 12-28 Var. 546 20-36 Var. 592 32-34 Var. 638 6-22 Var. 501 12-26 Var. 547 20-34 Var. 593 34-40 Var. 639 6-20 Var. 502 12-24 Var. 548 20-32 Var. 594 36-38 Var. 640 6-18 Var. 503 12-22 Var. 549 20-30 Var. 595 38-40 Var. 641

Var. = Option.

In some embodiments, a mat-rVWF composition produced by the methods presented herein can be characterized according to the percentage of mat-rVWF molecules that are present in a particular higher order mat-rVWF multimer or larger multimer. 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 an oligomeric complex consisting 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 an oligomeric complex consisting of at least 12 subunits. In other embodiments, the mat-rVWF composition used in the methods provided herein has a minimum percentage (e.g., has at least X%) of mat-rVWF molecules present in a particular higher order mat-rVWF multimer or larger multimer (for example, a multimer of at least Y subunits) according to any of options 134-457 shown in table. 5-7.

- 45 045553

Table 5 Exemplary embodiments of the percentage of mat-rVWF molecules that are present in a particular higher order mat-rVWF multimer or larger multimer found in the compositions and used in the methods presented herein

Minimum number of subunits in the rVWF multimer 6 8 10 12 14 16 Minimum percentage of rVWF molecules 10% Var. 134 Var. 152 Var. 170 Var. 188 Var. 206 Var. 224 15% Var. 135 Var. 153 Var. 171 Var. 189 Var. 207 Var. 225 20% Var. 136 Var. 154 Var. 172 Var. 190 Var. 208 Var. 226 25% Var. 137 Var. 155 Var. 173 Var. 191 Var. 209 Var. 227 thirty% Var. 138 Var. 156 Var. 174 Var. 192 Var. 210 Var. 228 35% Var. 139 Var. 157 Var. 175 Var. 193 Var. 211 Var. 229 40% Var. 140 Var. 158 Var. 176 Var. 194 Var. 212 Var. 230 45% Var. 141 Var. 159 Var. 177 Var. 195 Var. 213 Var. 231 50% Var. 142 Var. 160 Var. 178 Var. 196 Var. 214 Var. 232 55% Var. 143 Var. 161 Var. 179 Var. 197 Var. 215 Var. 233 60% Var. 144 Var. 162 Var. 180 Var. 198 Var. 216 Var. 234 65% Var. 145 Var. 163 Var. 181 Var. 199 Var. 217 Var. 235 70% Var. 146 Var. 164 Var. 182 Var. 200 Var. 218 Var. 236 75% Var. 147 Var. 165 Var. 183 Var. 201 Var. 219 Var. 237 80% Var. 148 Var. 166 Var. 184 Var. 202 Var. 220 Var. 238 85% Var. 149 Var. 167 Var. 185 Var. 203 Var. 221 Var. 239 90% Var. 150 Var. 168 Var. 186 Var. 204 Var. 222 Var. 240 95% Var. 151 Var. 169 Var. 187 Var. 205 Var. 223 Var. 241

Var. = Option.

Table 6 Exemplary embodiments of the percentage of mat-rVWF molecules that are present in a particular higher order mat-rVWF multimer or larger multimer found in the compositions and used in the methods presented herein

Minimum number of subunits in the rVWF multimer 18 20 22 24 26 28 i h s s 10% Var. 242 Var. 260 Var. 278 Var. 296 Var. 314 Var. 332 § | 15% Var. 243 Var. 261 Var. 279 Var. 297 Var. 315 Var. 333

- 46 045553

20% Var. 244 Var. 262 Var. 280 Var. 298 Var. 316 Var. 334 25% Var. 245 Var. 263 Var. 281 Var. 299 Var. 317 Var. 335 thirty% Var. 246 Var. 264 Var. 282 Var. 300 Var. 318 Var. 336 35% Var. 247 Var. 265 Var. 283 Var. 301 Var. 319 Var. 337 40% Var. 248 Var. 266 Var. 284 Var. 302 Var. 320 Var. 338 45% Var. 249 Var. 267 Var. 285 Var. 303 Var. 321 Var. 339 50% Var. 250 Var. 268 Var. 286 Var. 304 Var. 322 Var. 340 55% Var. 251 Var. 269 Var. 287 Var. 305 Var. 323 Var. 341 60% Var. 252 Var. 270 Var. 288 Var. 306 Var. 324 Var. 342 65% Var. 253 Var. 271 Var. 289 Var. 307 Var. 325 Var. 343 70% Var. 254 Var. 272 Var. 290 Var. 308 Var. 326 Var. 344 75% Var. 255 Var. 273 Var. 291 Var. 309 Var. 327 Var. 345 80% Var. 256 Var. 274 Var. 292 Var. 310 Var. 328 Var. 346 85% Var. 257 Var. 275 Var. 293 Var. 311 Var. 329 Var. 347 90% Var. 258 Var. 276 Var. 294 Var. 312 Var. 330 Var. 348 95% Var. 259 Var. 277 Var. 295 Var. 313 Var. 331 Var. 349

Var. = Option.

Table 7

Exemplary embodiments of the percentage of mat-rVWF molecules that are present in a particular higher order mat-rVWF multimer or larger multimer found in the compositions and used in the methods presented herein

Minimum number of subunits in the rVWF multimer thirty 32 34 36 38 40 Minimum percentage of rVWF molecules 10 % Var. 350 Var. 368 Var. 386 Var. 404 Var. 422 Var. 440 15 % Var. 351 Var. 369 Var. 387 Var. 405 Var. 423 Var. 441 20 % Var. 352 Var. 370 Var. 388 Var. 406 Var. 424 Var. 442 25 % Var. 353 Var. 371 Var. 389 Var. 407 Var. 425 Var. 443 thirty % Var. 354 Var. 372 Var. 390 Var. 408 Var. 426 Var. 444 35 % Var. 355 Var. 373 Var. 391 Var. 409 Var. 427 Var. 445

- 47 045553

40 % Var. 356 Var. 374 Var. 392 Var. 410 Var. 428 Var. 446 45 % Var. 357 Var. 375 Var. 393 Var. 411 Var. 429 Var. 447 50 % Var. 358 Var. 376 Var. 394 Var. 412 Var. 430 Var. 448 55 % Var. 359 Var. 377 Var. 395 Var. 413 Var. 431 Var. 449 60 % Var. 360 Var. 378 Var. 396 Var. 414 Var. 432 Var. 450 65 % Var. 361 Var. 379 Var. 397 Var. 415 Var. 433 Var. 451 70 % Var. 362 Var. 380 Var. 398 Var. 416 Var. 434 Var. 452 75 % Var. 363 Var. 381 Var. 399 Var. 417 Var. 435 Var. 453 80 % Var. 364 Var. 382 Var. 400 Var. 418 Var. 436 Var. 454 85 % Var. 365 Var. 383 Var. 401 Var. 419 Var. 437 Var. 455 90 % Var. 366 Var. 384 Var. 402 Var. 420 Var. 438 Var. 456 95 % Var. 367 Var. 385 Var. 403 Var. 421 Var. 439 Var. 457

Var. = Option.

Consistent with the above, mat-rVWF contains a significant percentage (HMW) of high molecular weight mat-rVWF multimers. In additional embodiments, the HMW rVWF multimer composition contains at least 10-80% mat-rVWF decamers or higher order multimers. In additional embodiments, the composition contains about 10-95%, 20-90%, 30-85%, 40-80%, 50-75%, 60-70% decamers or higher order multimers. In additional embodiments, the HMW mat-rVWF multimer composition contains at least about 10, 20, 30, 40, 50, 60, 70, 80, 90% decamers or higher order multimers.

Estimation of the amount and percentage of mat-rVWF multimers can be carried out using methods known in the art, including, without limitation, methods using electrophoresis and size exclusion chromatography methods for separating mat-rVWF by size, for example, as discussed in Cumming et al., (J Clin Pathol. 1993 May; 46(5): 470-473, which is incorporated herein by reference for all purposes and in particular for all recommendations relating to the evaluation of mat-rVWF multimers). Such methods may further include immunoblotting techniques (such as Western blotting) in which the gel is immunoblotted with a radiolabeled anti-VWF antibody followed by chemiluminescent detection (see, for example, Wen et al., (1993), J. Clin. Lab. Anal., 7: 317-323, which is incorporated herein by reference in its entirety for all purposes and in particular relevant to the evaluation of matrVWF multimers). Additional tests for VWF include VWF:antigen (VWF:Ag), VWF:ristocetin cofactor (VWF:RCof and VWF:collagen binding activity assay (VWF:CBA), which are often used to diagnose and classify von Willebrand disease (see e.g. , Favaloro et al., Pathology, 1997, 29(4): 341-456, which is hereby incorporated by reference in its entirety for all purposes and in particular for all techniques associated with VWF assays).

In some embodiments, the ratio of rFVIII procoagulant activity (ME rFVIII: C) to rVWF-ristocetin cofactor activity (ME rVWF: RCo) for mat-rVWF produced by the methods of this invention is from 3:1 to 1:5. In additional embodiments, the ratio is from 2:1 to 1:4. In other embodiments, the ratio is from 5:2 to 1:4. In additional embodiments, the ratio is from 3:2 to 1:3. In other countries

- 48 045553 In implementation options, 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 additional embodiments, the ratio is from 1:1 to 1:2. In other 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 some embodiments, the ratio of the procoagulant activity of rFVIII (ME rFVIII: C) to the activity of the ristocetin cofactor rVWF (ME rVWF: RCo) in the composition suitable for the method described herein is selected from options 1988 to 2140 listed in table. 8.

Table 8

Representative embodiments of the ratio of the procoagulant activity of rFVIII (ME rFVIII: C) to the activity of the ristocetin cofactor rVWF (ME rVWF: RCo) in the compositions and used in the methods proposed herein

(GO rFVin:C) to (IU rVWF:RCo) (GO rFVni:C) to (IU rVWF:RCo) (GO rFVin:C) to (IU rVWF:RCo) (GO rFVin:C) to (IU rVWF:RCo) 4:1 Var. 1988 3:1-3:5 Var. 2027 4:3-1:4 Var. 2065 4:5-2:3 Var. 2103

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3:1 Bap. 1989 3:1-2:3 Bap. 2028 4:3-1:3 Bap. 2066 4:5-3:4 Bap. 2104 2:1 Bap. 1990 3:1-3:4 Bap. 2029 4:3-2:5 Bap. 2067 3:4-1:6 Bap. 2105 3:2 Bap. 1991 3:1-4:5 Bap. 2030 4:3-1:2 Bap. 2068 3:4-1:5 Bap. 2106 4:3 Bap. 1992 3:1-5:6 Bap. 2031 4:3-3:5 Bap. 2069 3:4-1:4 Bap. 2107 1:1 Bap. 1993 3:1-1:1 Bap. 2032 4:3-2:3 Bap. 2070 3:4-1:3 Bap. 2108 5:6 Bap. 1994 3:1-4:3 Bap. 2033 4:3-3:4 Bap. 2071 3:4-2:5 Bap. 2109 4:5 Bap. 1995 3:1-3:2 Bap. 2034 4:3-4:5 Bap. 2072 3:4-1:2 Bap. 2110 3:4 Bap. 1996 3:1-2:1 Bap. 2035 4:3-5:6 Bap. 2073 3:4-3:5 Bap. 2111 2:3 Bap. 1997 2:1-1:6 Bap. 2036 4:3-1:1 Bap. 2074 3:4-2:3 Bap. 2112 3:5 Bap. 1998 2:1-1:5 Bap. 2037 1:1-1:6 Bap. 2075 2:3-1:6 Bap. 2113 1:2 Bap. 1999 2:1-1:4 Bap. 2038 1:1-1:5 Bap. 2076 2:3-1:5 Bap. 2114 2:5 Bap. 2000 2:1-1:3 Bap. 2039 1:1-1:4 Bap. 2077 2:3-1:4 Bap. 2115 1:3 Bap. 2001 2:1-2:5 Bap. 2040 1:1-1:3 Bap. 2078 2:3-1:3 Bap. 2116 1:4 Bap. 2002 2:1-1:2 Bap. 2041 1:1-2:5 Bap. 2079 2:3-2:5 Bap. 2117 1:5 Bap. 2003 2:1-3:5 Bap. 2042 1:1-1:2 Bap. 2080 2:3-1:2 Bap. 2118 1:6 Bap. 2004 2:1-2:3 Bap. 2043 1:1-3:5 Bap. 2081 2:3-3:5 Bap. 2119 4:1-1:6 Bap. 2005 2:1-3:4 Bap. 2044 1:1-2:3 Bap. 2082 3:5-1:6 Bap. 2120

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4:1-1:5 Var. 2006 2:1-4:5 Var. 2045 1:1-3:4 Var. 2083 3:5-1:5 Var. 2121 4:1-1:4 Var. 2007 2:1-5:6 Var. 2046 1:1-4:5 Var. 2084 3:5-1:4 Var. 2122 4:1-1:3 Var. 2008 2:1-1:1 Var. 2047 1:1-5:6 Var. 2085 3:5-1:3 Var. 2123 4:1-2:5 Var. 2009 2:1-4:3 Var. 2048 5:6-1:6 Var. 2086 3:5-2:5 Var. 2124 4:1-1:2 Var. 2010 2:1-3:2 Var. 2049 5:6-1:5 Var. 2087 3:5-1:2 Var. 2125 4:1-3:5 Var. 2011 3:2-1:6 Var. 2050 5:6-1:4 Var. 2088 1:2-1:6 Var. 2126 4:1-2:3 Var. 2012 3:2-1:5 Var. 2051 5:6-1:3 Var. 2089 1:2-1:5 Var. 2127 4:1-3:4 Var. 2013 3:2-1:4 Var. 2052 5:6-2:5 Var. 2090 1:2-1:4 Var. 2128 4:1-4:5 Var. 2014 3:2-1:3 Var. 2053 5:6-1:2 Var. 2091 1:2-1:3 Var. 2129 4:1-5:6 Var. 2015 3:2-2:5 Var. 2054 5:6-3:5 Var. 2092 1:2-2:5 Var. 2130 4:1-1:1 Var. 2016 3:2-1:2 Var. 2055 5:6-2:3 Var. 2093 2:5-1:6 Var. 2131 4:1-4:3 Var. 2017 3:2-3:5 Var. 2056 5:6-3:4 Var. 2094 2:5-1:5 Var. 2132 4:1-3:2 Var. 2018 3:2-2:3 Var. 2057 5:6-4:5 Var. 2095 2:5-1:4 Var. 2133 4:1-2:1 Var. 2019 3:2-3:4 Var. 2058 4:5-1:6 Var. 2096 2:5-1:3 Var. 2134 4:1-3:1 Var. 2020 3:2-4:5 Var. 2059 4:5-1:5 Var. 2097 1:3-1:6 Var. 2135 3:1-1:6 Var. 2021 3:2-5:6 Var. 2060 4:5-1:4 Var. 2098 1:3-1:5 Var. 2136 3:1-1:5 Var. 2022 3:2-1:1 Var. 2061 4:5-1:3 Var. 2099 1:3-1:4 Var. 2137 3:1-1:4 Var. 2023 3:2-4:3 Var. 2062 4:5-2:5 Var. 2100 1:4-1:6 Var. 2138 3:1-1:3 Var. 2024 4:3-1:6 Var. 2063 4:5-1:2 Var. 2101 1:4-1:5 Var. 2139 3:1-2:5 Var. 2025 4:3-1:5 Var. 2064 4:5-3:5 Var. 2102 1:5-1:6 Var. 2140 3:1-1:2 Var. 2026

Var. = Option.

In additional embodiments, the higher order mat-rVWF multimers of this invention are stable from about 1 to about 90 hours after administration. In other embodiments, higher order mat-rVWF multimers are stable for about 5-80, 10-70,

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15-60, 20-50, 25-40, 30-35 hours after administration. In other embodiments, higher order mat-rVWF multimers are stable for at least 3, 6, 12, 18, 24, 36, 48, 72 hours after administration. In some embodiments, the in vitro stability of mat-rVWF multimers is assessed.

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, higher order mat-rVWF multimers have a half-life of at least 24 hours after administration. In other embodiments, higher order mat-rVWF multimers have a half-life selected from options 642 to 1045 listed in Table. 9.

Table 9

Exemplary embodiments of the half-life of higher order mat-rVWF multimers found in compositions prepared by the methods proposed herein

Watch Watch Watch Watch at least 1 Var. 642 4-22 Var. 743 14-78 Var. 844 24-30 Var. 945 at least 2 Var. 643 4-20 Var. 744 14-72 Var. 845 24-27 Var. 946 at least 3 Var. 644 4-18 Var. 745 14-66 Var. 846 27-90 Var. 947 at least 4 Var. 645 4-16 Var. 746 14-60 Var. 847 27-84 Var. 948 at least 5 Var. 646 4-14 Var. 747 14-54 Var. 848 27-78 Var. 949 at least 6 Var. 647 4-12 Var. 748 14-48 Var. 849 27-72 Var. 950 at least 7 Var. 648 4-10 Var. 749 14-45 Var. 850 27-66 Var. 951 at least 8 Var. 649 4-8 Var. 750 14-42 Var. 851 27-60 Var. 952 at least 9 Var. 650 4-6 Var. 751 14-39 Var. 852 27-54 Var. 953

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at least 10 Var. 651 6-90 Var. 752 14-36 Var. 853 27-48 Var. 954 at least 11 Var. 652 6-84 Var. 753 14-33 Var. 854 30-90 Var. 955 at least 12 Var. 653 6-78 Var. 754 14-30 Var. 855 30-84 Var. 956 at least 14 Var. 654 6-72 Var. 755 14-27 Var. 856 30-78 Var. 957 at least 16 Var. 655 6-66 Var. 756 14-24 Var. 857 30-72 Var. 958 at least 18 Var. 656 6-60 Var. 757 14-22 Var. 858 30-66 Var. 959 at least 20 Var. 657 6-54 Var. 758 14-20 Var. 859 30-60 Var. 960 at least 22 Var. 658 6-48 Var. 759 14-18 Var. 860 30-54 Var. 961 at least 24 Var. 659 6-45 Var. 760 14-16 Var. 861 30-48 Var. 962 at least 27 Var. 660 6-42 Var. 761 16-90 Var. 862 30-45 Var. 963 at least 30 Var. 661 6-39 Var. 762 16-84 Var. 863 30-42 Var. 964 at least 33 Var. 662 6-36 Var. 763 16-78 Var. 864 30-39 Var. 965 at least 36 Var. 663 6-33 Var. 764 16-72 Var. 865 30-36 Var. 966 at least 39 Var. 664 6-30 Var. 765 16-66 Var. 866 30-33 Var. 967 at least 42 Var. 665 6-27 Var. 766 16-60 Var. 867 33-90 Var. 968 at least 45 Var. 666 6-24 Var. 767 16-54 Var. 868 33-84 Var. 969 at least 48 Var. 667 6-22 Var. 768 16-48 Var. 869 33-78 Var. 970 at least 54 Var. 668 6-20 Var. 769 16-45 Var. 870 33-72 Var. 971 at least 60 Var. 669 6-18 Var. 770 16-42 Var. 871 33-66 Var. 972 at least 66 Var. 670 6-16 Var. 771 16-39 Var. 872 33-60 Var. 973 at least 72 Var. 671 6-14 Var. 772 16-36 Var. 873 33-54 Var. 974 at least 78 Var. 672 6-12 Var. 773 16-33 Var. 874 33-48 Var. 975 at least 84 Var. 673 6-10 Var. 774 16-30 Var. 875 33-45 Var. 976 at least 90 Var. 674 6-8 Var. 775 16-27 Var. 876 33-42 Var. 977 2-90 Var. 675 8-90 Var. 776 16-24 Var. 877 33-29 Var. 978 2-84 Var. 676 8-84 Var. 777 16-22 Var. 878 33-36 Var. 979 2-78 Var. 677 8-78 Var. 778 16-20 Var. 879 36-90 Var. 980 2-72 Var. 678 8-72 Var. 779 16-18 Var. 880 36-84 Var. 981 2-66 Var. 679 8-66 Var. 780 18-90 Var. 881 36-78 Var. 982 2-60 Var. 680 8-60 Var. 781 18-84 Var. 882 36-72 Var. 983 2-54 Var. 681 8-54 Var. 782 18-78 Var. 883 36-66 Var. 984 2-48 Var. 682 8-48 Var. 783 18-72 Var. 884 36-60 Var. 985 2-45 Var. 683 8-45 Var. 784 18-66 Var. 885 36-54 Var. 986 2-42 Var. 684 8-42 Var. 785 18-60 Var. 886 36-48 Var. 987

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2-39 Bap. 685 8-39 Bap. 786 18-54 Bap. 887 36-45 Bap. 988 2-36 Bap. 686 8-36 Bap. 787 18-48 Bap. 888 36-42 Bap. 989 2-33 Bap. 687 8-33 Bap. 788 18-45 Bap. 889 36-39 Bap. 990 2-30 Bap. 688 8-30 Bap. 789 18-42 Bap. 890 39-90 Bap. 991 2-27 Bap. 689 8-27 Bap. 790 18-39 Bap. 891 39-84 Bap. 992 2-24 Bap. 690 8-24 Bap. 791 18-36 Bap. 892 39-78 Bap. 993 2-22 Bap. 691 8-22 Bap. 792 18-33 Bap. 893 39-72 Bap. 994 2-20 Bap. 692 8-20 Bap. 793 18-30 Bap. 894 39-66 Bap. 995 2-18 Bap. 693 8-18 Bap. 794 18-27 Bap. 895 39-60 Bap. 996 2-16 Bap. 694 8-16 Bap. 795 18-24 Bap. 896 39-54 Bap. 997 2-14 Bap. 695 8-14 Bap. 796 18-22 Bap. 897 39-48 Bap. 998 2-12 Bap. 696 8-12 Bap. 797 18-20 Bap. 898 39-45 Bap. 999 2-10 Bap. 697 8-10 Bap. 798 20-90 Bap. 899 39-42 Bap. 1000 2-8 Bap. 698 10-90 Bap. 799 20-84 Bap. 900 42-90 Bap. 1001 2-6 Bap. 699 10-84 Bap. 800 20-78 Bap. 901 42-84 Bap. 1002 2-4 Bap. 700 10-78 Bap. 801 20-72 Bap. 902 42-78 Bap. 1003 3-90 Bap. 701 10-72 Bap. 802 20-66 Bap. 903 42-72 Bap. 1004 3-84 Bap. 702 10-66 Bap. 803 20-60 Bap. 904 42-66 Bap. 1005 3-78 Bap. 703 10-60 Bap. 804 20-54 Bap. 905 42-60 Bap. 1006 3-72 Bap. 704 10-54 Bap. 805 20-48 Bap. 906 42-54 Bap. 1007 3-66 Bap. 705 10-48 Bap. 806 20-45 Bap. 907 42-48 Bap. 1008 3-60 Bap. 706 10-45 Bap. 807 20-42 Bap. 908 42-45 Bap. 1009 3-54 Bap. 707 10-42 Bap. 808 20-39 Bap. 909 45-90 Bap. 1010 3-48 Bap. 708 10-39 Bap. 809 20-36 Bap. 910 45-84 Bap. 1011 3-45 Bap. 709 10-36 Bap. 810 20-33 Bap. 911 45-78 Bap. 1012 3-42 Bap. 710 10-33 Bap. 811 20-30 Bap. 912 45-72 Bap. 1013 3-39 Bap. 711 10-30 Bap. 812 20-27 Bap. 913 45-66 Bap. 1014 3-36 Bap. 712 10-27 Bap. 813 20-24 Bap. 914 45-60 Bap. 1015 3-33 Bap. 713 10-24 Bap. 814 20-22 Bap. 915 45-54 Bap. 1016 3-30 Bap. 714 10-22 Bap. 815 22-90 Bap. 916 45-48 Bap. 1017 3-27 Bap. 715 10-20 Bap. 816 22-84 Bap. 917 48-90 Bap. 1018 3-24 Bap. 716 10-18 Bap. 817 22-78 Bap. 918 48-84 Bap. 1019 3-22 Bap. 717 10-16 Bap. 818 22-72 Bap. 919 48-78 Bap. 1020 3-20 Bap. 718 10-14 Bap. 819 22-66 Bap. 920 48-72 Bap. 1021

- 54 045553

3-18 Bap. 719 10-12 Bap. 820 22-60 Bap. 921 48-66 Bap. 1022 3-16 Bap. 720 12-90 Bap. 821 22-54 Bap. 922 48-60 Bap. 1023 3-14 Bap. 721 12-84 Bap. 822 22-48 Bap. 923 48-54 Bap. 1024 3-12 Bap. 722 12-78 Bap. 823 22-45 Bap. 924 54-90 Bap. 1025 3-10 Bap. 723 12-72 Bap. 824 22-42 Bap. 925 54-84 Bap. 1026 3-8 Bap. 724 12-66 Bap. 825 22-39 Bap. 926 54-78 Bap. 1027 3-6 Bap. 725 12-60 Bap. 826 22-36 Bap. 927 54-72 Bap. 1028 3-4 Bap. 726 12-54 Bap. 827 22-33 Bap. 928 54-66 Bap. 1029 4-90 Bap. 727 12-48 Bap. 828 22-30 Bap. 929 54-60 Bap. 1030 4-84 Bap. 728 12-45 Bap. 829 22-27 Bap. 930 60-90 Bap. 1031 4-78 Bap. 729 12-42 Bap. 830 22-24 Bap. 931 60-84 Bap. 1032 4-72 Bap. 730 12-39 Bap. 831 24-90 Bap. 932 60-78 Bap. 1033 4-66 Bap. 731 12-36 Bap. 832 24-84 Bap. 933 60-72 Bap. 1034 4-60 Bap. 732 12-33 Bap. 833 24-78 Bap. 934 60-66 Bap. 1035 4-54 Bap. 733 12-30 Bap. 834 24-72 Bap. 935 66-90 Bap. 1036 4-48 Bap. 734 12-27 Bap. 835 24-66 Bap. 936 66-84 Bap. 1037 4-45 Bap. 735 12-24 Bap. 836 24-60 Bap. 937 66-78 Bap. 1038 4-42 Bap. 736 12-22 Bap. 837 24-54 Bap. 938 66-72 Bap. 1039 4-39 Bap. 737 12-20 Bap. 838 24-48 Bap. 939 72-90 Bap. 1040 4-36 Bap. 738 12-18 Bap. 839 24-45 Bap. 940 72-84 Bap. 1041 4-33 Bap. 739 12-16 Bap. 840 24-42 Bap. 941 72-78 Bap. 1042 4-30 Bap. 740 12-14 Bap. 841 24-39 Bap. 942 78-90 Bap. 1043 4-27 Bap. 741 14-90 Bap. 842 24-36 Bap. 943 78-84 Bap. 1044 4-24 Bap. 742 14-84 Bap. 843 24-33 Bap. 944 84-90 Bap. 1045

Var. = Option.

In some embodiments, pro-VWF and/or purified mat-rVWF purified in accordance with this invention is not modified by any conjugation, post-translation, or covalent modifications. In specific embodiments, the pro-VWF and/or purified mat-rVWF of this invention is not modified with a water-soluble polymer, including, without limitation, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylene, polysialic acid, hydroxyethyl starch, polycarbohydrate moiety, and the like.

In some embodiments, pro-VWF and/or purified mat-rVWF purified in accordance with the present invention is modified by conjugation, post-translational modification, or covalent modification, including modifications of N- or C-terminal residues, as well as modifications of selected side chains, e.g. , at free sulfhydryl groups, primary amines and hydroxyl groups. In one embodiment, the water-soluble polymer is linked to the protein (directly or via a linker) by a lysine group or other primary amine. In some embodiments, pro-VWF and/or purified mat-rVWF of this 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, and the like .

Water-soluble polymers that can be used to modify pro-VWF and/or purified mat-rVWF include linear and branched structures. Conjugated polymers can be attached directly to the coagulation proteins of the invention or, alternatively, can be attached via a linking moiety. Non-limiting examples of protein conjugation to water-soluble polymers can be found in US Pat. No. 4,640,835; 4496689; 4301144; 4670417; 4791192 and 4179337, and in Abuchowski and Davis Enzymes as Drugs, Holcenberg and Roberts, Eds., pp. 367 383, John Wiley and Sons, New York (1981), and Hermanson G., Bioconjugate Techniques 2nd Ed., Academic Press, Inc. 2008.

Protein conjugation can be accomplished using a number of techniques well known in the art, for example, see G. Hermanson, Bioconjugate Techniques 2nd Ed., Academic Press, Inc. 2008. Examples include communication via a peptide bond between a carboxyl group on one of the koa proteins

- 55 045553 gulation or water-soluble polymer fragment and the amino group of the other, or an ester bond between the carboxyl group of one and the hydroxyl group of the other. Another linkage by which the coagulation protein of this invention can be conjugated to a water-soluble polymer compound is through a Schiff base between a free amino group on the polymer moiety that reacts with an aldehyde group formed on the non-reducing end of the polymer by periodate oxidation (Jennings and Lugowski, J Immunol 1981;127:10118; Femandes and Gregonradis, Biochim Biophys Acta 1997;1341;26-34). The resulting Schiff base can be stabilized by specific reduction with NaCNBH ₃ to form a secondary amine. An alternative approach is to obtain terminal free amino groups on the polymer by reductive amination with NH ₄ Cl after preliminary oxidation. Bifunctional reagents can be used to link two amino or two hydroxyl groups. For example, a polymer containing an amino group can be coupled to the amino group of a coagulation protein using reagents such as BS3 (bis(sulfosuccinimidyl)suberate/Pierce, Rockford, IL, USA). In addition, heterobifunctional cross-linking reagents such as sulfo-EMCS (N-εmaleimidocaproyloxy) sulfosuccinimide ester/Pierce) can be used, for example, to couple amine and thiol groups. In other embodiments, an aldehyde-reactive group such as a PEG alkoxide plus bromoacetaldehyde diethyl acetal; PEG plus DMSO and acetic anhydride, as well as PEG chloride plus 4-hydroxybenzaldehyde phenoxide, succinimidyl active esters, activated PEG dithiocarbonate, 2,4,5-trichlorophenyl chloroformate and PEG activated P-nitrophenyl chloroformate can be used in coagulation protein conjugation.

Another method for measuring the biological activity of VWF is a collagen binding assay based on ELISA technology (Brown and Bosak, Thromb. Res., 1986, 43:303-311; Favaloro, Thromb. Haemost., 2000, 83 127-135). The microtiter plate is coated with collagen type I or III. VWF then binds to the collagen surface and is subsequently detected by an enzyme-labeled polyclonal antibody. The last step is the substrate reaction, which can be photometrically monitored using an ELISA reader.

Von Willebrand factor immunoassays (VWF:Ag) are immunoassays that measure the concentration of VWF protein in plasma. They do not point to the VWF function. There are a number of methods for measuring VWF:Ag, including both enzyme-linked immunosorbent assay (ELISA) and automated enzyme-linked immunosorbent assay (ELIA). Many laboratories now use fully automated enzyme immunoassays. Historically, laboratories have used a variety of methods, including the Laurell Laurell Rockets electroimmunoassay, but these are rarely used in most laboratories today.

Compositions/Introduction VWF

The method of this invention also provides for the preparation of VWF compositions obtained by the purification methods presented herein. In some embodiments, the highly purified mat-rVWF composition is used to produce a pharmaceutical composition. In some embodiments, mat-rVWF may be included in a lyophilized formulation.

In some embodiments, formulations containing the VWF polypeptide of the invention are lyophilized after purification and before administration to a subject. Lyophilization is carried out using methods generally accepted in the art and must be optimized for the formulation being developed (Tang et al., Pharm Res. 21:191-200, (2004) and Chang et al., Pharm Res. 13:243-9 (1996 )).

The lyophilization cycle, in one aspect, consists of three stages: freezing, primary drying and secondary drying (A. R. Mackenzie, Phil Trans R Soc London, Ser B, Biol 278:167 (1977)). During the freezing stage, the solution is cooled to cause ice to form. In addition, this step causes crystallization of the bulking agent. Ice is sublimated in the primary drying stage, which is carried out by reducing the pressure in the chamber below the vapor pressure of the ice, using vacuum and heat to accelerate sublimation. Finally, the adsorbed or bound water is removed in a secondary drying step under reduced chamber pressure and elevated shelf temperature. The result is a material known as freeze-dried cake. The cake can then be reconstituted with sterile water or a suitable injection diluent.

The lyophilization cycle not only determines the final physical state of the excipients, but also influences other parameters such as reconstitution time, appearance, stability and final moisture content. The structure of the composition in the frozen state goes through several transitions (eg, glass transition, wetting, and crystallization) that occur at certain temperatures, and this structure can 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 determined using differential scanning calorimetry (DSC). Tg and Tg' are important parameters to consider when designing a lyophilization cycle. For example, Tg' is important for primary drying. In addition, when dried, tempo

- 56 045553 The glass transition temperature provides information about the storage temperature of the final product.

Pharmaceutical compositions and excipients in general.

Excipients are additives that either impart or improve the stability and delivery of a drug product (such as a protein). Regardless of the reason for their inclusion, excipients are an integral component of the drug and therefore should be safe and well tolerated by patients. For protein drugs, the choice of excipients is especially important because they can influence both the efficacy and immunogenicity of the drug. Therefore, protein formulations need to be formulated with appropriate selection of excipients that provide suitable stability, safety and marketability.

The lyophilized composition, in one aspect, is at least composed of one or more of a buffer, a bulking agent, and a stabilizer. In this aspect, the usefulness of the surfactant is evaluated and selected in cases where aggregation during the lyophilization step or during reconstitution becomes a problem. An appropriate buffering agent is included to maintain the formulation within a stable pH range during lyophilization. A comparison of the excipient components provided for liquid and lyophilized protein formulations is presented in Table. 10.

Table 10

Excipient components of lyophilized protein compositions

Excipient component Function in lyophilized formulation Buffer Maintains formulation pH during lyophilization and after reconstitution Tonicity regulator/stabilizer Stabilizers include cryoprotectors and lyoprotectors. Examples include polyols, sugars and polymers. Cryoprotectors protect proteins from cold stress Lyoprotectors stabilize proteins in a lyophilized state Volume-forming agent Used to improve product uniformity and prevent blowout Provides structural strength to lyo-sludge Examples include mannitol and glycine. Surface active substance Used when aggregation during lyophilization is a problem May help reduce recovery time Examples include polysorbate 20 and 80 Antioxidant Not usually used, molecular reactions in the lyo-sediment are significantly slowed down. Metal ions/chelating agent May be included if a particular metal ion is included only as a cofactor or if the metal is required for protease activity Chelating agents are not usually required in lyo formulations Preservative For multi-dose formulations only Provides protection against microbial growth in formulation Typically included in reconstitution diluent (e.g. bWFI)

A major challenge in developing protein formulations is to stabilize the product against the stresses associated with production, transportation and storage. The role of the excipient in the formulation is to provide stabilization against these stresses. Excipients are also used to reduce the viscosity of high-concentration protein formulations to aid delivery and improve patient convenience. In general, excipients can be classified based on the mechanisms by which they stabilize proteins against various chemical and physical stresses. Some excipients are used to mitigate the effects of a particular stress or to regulate a particular susceptibility to a particular protein. Other excipients have a more general effect on the physical and covalent stability of proteins. The excipients described in this document are grouped by chemical type or functional role in the formulations. A brief description of stabilization modes is provided when discussing each type of excipient.

Given the ideas and guidelines presented herein, those skilled in the art will know what amount or range of excipient can be included in any particular formulation to formulate the biopharmaceutical composition of the invention that helps maintain the stability of the biopharmaceutical composition (eg, protein). For example, the amount and type of salt to be included in the biopharmaceutical composition of the present invention is selected based on the desired osmolality (eg, isotonic, hypotonic, or hypertonic) of the final solution, as well as the amount and osmolality of other components to be included in the formulation.

For example, inclusion of about 5% sorbitol can provide isotonicity, while about 9% sucrose excipient is required to achieve isotonicity. The choice of the amount or range of concentrations of one or more excipients that may be included in the biopharmaceutical composition of this invention has been illustrated above with reference to salts, polyols and sugars. However, those skilled in the art will appreciate that the considerations described herein and further illustrated by reference to specific excipients apply equally to all types and combinations of excipients, including, for example, salts, amino acids, other tonicity agents, surfactants , stabilizers, bulking agents, cryoprotectants, lyoprotectants, antioxidants, metal ions, chelating agents and/or preservatives.

In addition, if a particular excipient is specified in molar concentration, those skilled in the art will understand that the equivalent percentage of solution (%) w/v is also considered. (e.g. (grams of substance in sample solution/ml of solution) X 100%).

Of course, one skilled in the art will appreciate that the concentrations of the excipients described herein are interdependent within a particular formulation. For example, the concentration of the bulking agent may be reduced where, for example, there is a high concentration of protein or where, for example, there is a high concentration of a stabilizing agent. Additionally, one skilled in the art will appreciate that to maintain isotonicity of a particular formulation that does not contain a bulking agent, the concentration of the stabilizing agent must be adjusted accordingly (eg, a tonic amount of stabilizer will be used). Common excipients are known in the art and can be found in Powell et al., Compendium of Excipients fir Parenteral Formulations (1998), PDA J. Pharm. Sci. Technology, 52:238-311.

Pharmaceutical buffers and buffering agents.

Typically, maximum stability of the pharmacologically active protein composition is observed in a narrow pH range. This range of optimal stability must be determined early in the research before formulation. Several approaches, such as accelerated stability studies and calorimetric screening studies, are useful in these applications (Remmele R. L. Jr., et al., Biochemistry, 38(16): 5241-7 (1999)). Once the formulation is ready, the protein must be produced and preserved throughout its shelf life. Consequently, buffering agents are almost always used to control the pH of a formulation.

The buffering capacity of buffer substances is maximum at a pH equal to pKa and decreases as the pH increases or decreases from this value. Ninety percent of the buffer capacity is within one pH unit of its pKa. The buffer capacity also increases in proportion to the increase in buffer concentration.

There are several factors to consider when choosing a buffer. First of all, it is necessary to determine the type of buffer and its concentration based on its pKa and the desired pH of the drug. It is equally important to ensure that the buffer is compatible with the protein and other excipients of the composition and does not catalyze any degradation reactions. The third important aspect to consider is the tingling sensation and irritation that the buffer may cause upon injection. For example, citrate is known to cause a tingling sensation when injected (Laursen T, et al., Basic Clin Pharmacol Toxicol., 98(2): 218-21 (2006)). The potential for tingling and irritation is greater for drugs that are administered by the subcutaneous (SC) or intramuscular (IM) routes, where the drug solution remains in place for a relatively longer period of time than with intravenous administration, where the formulation dissolves quickly in the blood upon administration . For formulations that are administered by direct intravenous infusion, the total amount of buffer (and any other component of the formulation) must be monitored. Particular caution should be exercised with potassium ions administered in the form of potassium phosphate buffer, which may cause cardiovascular effects in the patient (Hollander-Rodriguez JC, et al., Am. Fam. Physician., 73(2): 283-90 (2006)).

Buffers for lyophilized formulations require additional consideration. Some buffers, such as sodium phosphate, can crystallize from the amorphous protein phase during freezing, resulting in a pH shift. Other common buffers, such as acetate and imidazole, may sublimate or evaporate during the lyophilization process, thereby changing the pH of the composition

- 58 045553 during lyophilization or after reconstitution.

The buffer system present in the compositions is selected to be physiologically compatible and to maintain the desired pH of the pharmaceutical composition. In one embodiment, the pH of the solution is between pH 2.0 and pH 12.0. For example, the pH of a solution can 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.

The pH control buffer compound may be present in any amount suitable to maintain the pH of the composition at a desired level. In one embodiment, the concentration of the pH control compound is from 0.1 mM to 500 mM (1 M). For example, it is contemplated that the concentration of the pH control compound is 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.

Examples of pH control agents used to buffer the formulation set forth 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 restrictions, aspartate, histidine and glycine. In one embodiment of this invention, the buffering agent is citrate.

In some embodiments, the composition contains 50 mM glycine, 10 mM taurine, 5% (w/w) sucrose, 5% (w/w) D-mannitol, 0.1% polysorbate 80, 2 mM CaCl ₂ , 150 mM NaCl and has a pH of 7.4. In some embodiments, the formulation contains high purity mat-rVWF, 50 mM glycine, 10 mM taurine, 5% (w/w) sucrose, 5% (w/w) D-mannitol, 0.1% polysorbate 80 , 2 mM CaCl ₂ , 150 mM NaCl and has a pH of 7.4. In some embodiments, the formulation contains 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. CaCl ₂ , 150 mM NaCl and pH 7.4.

Pharmaceutical stabilizers and bulking agents.

In one aspect of these pharmaceutical formulations, a stabilizer (or combination of stabilizers) is added to prevent or reduce aggregation and chemical degradation caused by storage. A cloudy or opaque solution after reduction indicates that the protein has precipitated or at least aggregated. The term stabilizer means an excipient capable of preventing aggregation or physical degradation, including chemical degradation (eg, autolysis, deamidation, oxidation, etc.) in an aqueous state. Suspected stabilizers include, but are not limited to, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, glycine, arginine HCL, polyhydroxy compounds including polysaccharides such as dextran, starch, hydroxyethyl starch, cyclodextrins, N-methylpyrollidene, cellulose and hyaluronic acid, sodium chloride (Carpenter et al., Develop. Biol. Standard 74:225, (1991)). In these compositions, the stabilizer is included in concentrations 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 present invention, mannitol and trehalose are used as stabilizing agents.

If desired, the formulations also include appropriate amounts of volume and osmolality controlling agents. Bulking agents include, for example, 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 introduced in concentrations 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.

Pharmaceutical surfactants.

Proteins have a high propensity to interact with surfaces, making them susceptible to adsorption and denaturation at air-liquid, bubble-liquid, and liquid-liquid (silicon grease) interfaces. This degradation pathway was found to be inversely dependent on protein concentration and resulted in either the formation of soluble and insoluble protein aggregates or loss of protein from solution through adsorption to surfaces. In addition to adsorption to the surface of the container, degradation caused by the surface is exacerbated by physical agitation, such as during transport and handling of the product.

Surfactants are commonly used in protein formulations to prevent surface-induced degradation. Surfactants are amphipathic molecules that can displace proteins at interfacial positions. The hydrophobic portions of the surfactant molecules occupy interfacial positions (e.g., air/liquid), while the hydrophilic portions of the molecules remain oriented toward the bulk solvent. At sufficient concentrations (usually around the critical micellar detergent concentration), a surface layer of surfactant molecules serves to prevent the adsorption of protein molecules at the interface. Thus, degradation caused by the surface is reduced to a minimum. Surfactants contemplated herein include, but are not limited to, fatty acid esters of sorbitan polyethoxylates, such as polysorbate 20 and polysorbate 80. They differ only in the length of the aliphatic chain, which imparts the hydrophobic character to the C-12 and C-18 molecules, respectively. Accordingly, polysorbate-80 is more surface active and has a lower critical micellar concentration than polysorbate-20.

Detergents can also affect the thermodynamic conformational stability of proteins. Here again the effects of a given detergent excipient will be protein specific. For example, polysorbates have been shown to reduce the stability of some proteins and increase the stability of others. Destabilization of proteins by detergent can be explained in terms of the hydrophobic tails of detergent molecules, which may be involved in specific binding to partially or fully unfolded states of the protein. These types of interactions can cause a shift in conformational equilibrium toward more expanded states of the protein (e.g., increasing exposure of hydrophobic portions of the protein molecule in addition to the polysorbate binder). Alternatively, if the protein's native state exhibits some hydrophobic surfaces, detergent binding to the native state may stabilize this conformation.

Another aspect of polysorbates is that they are inherently susceptible to oxidative damage. They often contain sufficient peroxides as raw materials to cause oxidation of the side chains of protein residues, especially methionine. The potential for oxidative damage arising from the addition of a stabilizer emphasizes that the lowest effective concentrations of excipients should be used in formulations. For surfactants, the effective concentration of a given protein will depend on the stabilization mechanism.

Surfactants are also added in appropriate amounts to prevent the phenomenon of aggregation on the surface during freezing and drying (Chang, B, J. Pharm. Sci. 85:1325, (1996)). Thus, typical surfactants include, but are not limited to, anionic, cationic, nonionic, zwitterionic and amphoteric surfactants, including surfactants derived from naturally occurring amino acids. Anionic surfactants include, but are not limited to, sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate, chenodeoxycholic acid, sodium N-lauroyl sarcosine, lithium dodecyl sulfate, sodium 1-octane sulfonic acid, sodium cholate hydrate, sodium deoxycholate and sodium glycodeoxycholic acid. 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, TWEEN20 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, soy lecithin and other phospholipids such as dioleylphosphatidylcholine . (DOPC), dimyristoylphosphatidylglycerol (DMPG), dimyristoylphosphatidylcholine (DMPC) and (dioleylphosphatidylglycerol) DOPG; sucrose fatty acid ester, methylcellulose and carboxymethylcellulose. Therefore, compositions containing these surfactants either individually or as a mixture in various ratios are additionally provided. In one embodiment of this invention, the surfactant is TWEEN-80. In these formulations, the surfactant is added at a concentration of about 0.01 to about 0.5 g/L. In the proposed compositions, 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.

Pharmaceutical salts

Salts are often added to increase the ionic strength of the drug, which can be important for protein solubility, physical stability, and isotonicity. Salts can affect the physical stability of proteins in different ways. Ions can stabilize the natural state of proteins by binding to charged residues on the surface of the protein. 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 in the protein molecule. Salts in protein formulations can also protect attractive electrostatic interactions between protein molecules, which can lead to protein aggregation and insolubility. In the proposed formulations, the salt concentration is between 0.1, 1, 10, 20, 30, 40, 50, 8θ, 100, 120, 150, 200, 300 and 500 mM.

Other common excipient components: Pharmaceutical amino acids.

Amino acids have found universal use in protein formulations as buffers, bulking agents, stabilizers and antioxidants. Thus, in one aspect, histidine and glutamic acid are used to buffer protein compositions in the pH range 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 side chain group of glutamic acid has a pKa of 4.3, making these amino acids suitable for buffering in their

- 60 045553 corresponding pH ranges. In such cases, glutamic acid is especially useful. Histidine is commonly found in commercial protein products, and this amino acid provides an alternative to citrate, a buffer that is known to cause a burning sensation when injected. Interestingly, histidine also has a stabilizing effect on aggregation when used at high concentrations in both liquid and lyophilized formulations (Chen B, et al., Pharm Res., 20(12): 1952-60 (2003)). Others have observed that histidine reduces the viscosity of a high protein concentration drug. However, in the same study, the authors observed increased aggregation and discoloration of formulations containing histidine during antibody studies under freeze-thaw conditions in stainless steel containers. Another caveat with the use of histidine is that it undergoes photo-oxidation 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 a number of oxidative stresses (Lam XM, et al., J Pharm ScL, 86(11): 1250-5 (1997)).

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

In the proposed compositions, the concentration of amino acids is 0.1, 1, 10, 20, 30, 40, 50, 80, 100, 120, 150, 200, 300 and 500 mM. In one embodiment of this invention, the amino acid is glycine.

Other common excipient components: Pharmaceutical antioxidants.

Oxidation of protein residues occurs from various sources. In addition to adding specific antioxidants, preventing oxidative damage to proteins involves careful control of a number of factors during product production and storage, such as atmospheric oxygen, temperature, light exposure, and chemical contamination. Therefore, the invention contemplates the use of pharmaceutical antioxidants, including, without limitation, reducing agents, oxygen/free radical scavengers, or chelating agents. Antioxidants in therapeutic protein compositions in one aspect are water soluble and remain active throughout the shelf life of the product. Reducing agents and oxygen/free radical scavengers work by removing reactive oxygen species in solution. Chelating agents such as EDTA are effective by binding trace metal impurities that contribute to the formation of free radicals. For example, EDTA has been used in a liquid formulation of acidic fibroblast growth factor to inhibit the oxidation of cysteine residues catalyzed by metal ions.

In addition to the effectiveness of various excipients in preventing protein oxidation is the ability of the antioxidants themselves to cause other covalent or physical changes to the protein. For example, reducing agents can cause disruption of intramolecular disulfide bonds, which can lead to disulfide shuffling. In the presence of transition metal ions, ascorbic acid and EDTA have been shown to promote the oxidation of methionine in a number of 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 J.R., /. 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 light- and temperature-induced methionine oxidation levels in rhuMab HER2; however, this study also reported the formation of a thiosulfate-protein adduct (Lam XM, Yang JY, et al., J Pharm Sci. 86(11): 1250-5 (1997)). The choice of a suitable antioxidant is made depending on the specific loads and protein sensitivity. Antioxidants contemplated in certain aspects include, but are not limited to, reducing agents and oxygen/free radical scavengers, EDTA and sodium thiosulfate.

Other common excipient components: Pharmaceutical metal ions.

In general, transition metal ions are undesirable in protein formulations because they can catalyze physical and chemical degradation reactions of proteins. However, specific metal ions are included in formulations when they are cofactors of proteins, and in protein suspension formulations where they form coordination complexes (eg, zinc suspension of insulin). Recently, the use of magnesium ions (10-120 mM) has been proposed to inhibit the isomerization of aspartic acid to isoaspartic acid (WO 2004039337).

Two examples in which metal ions confer stability or increased activity on proteins are human deoxyribonuclease (rhDNase, Pulmozyme®) and factor VIII. In the case of rhDNase, Ca ²⁺ ions (up to 100 mM) increase the stability of the enzyme through a specific binding site. (Chen B, et al., / Pharm Sci., 88(4): 477-82 (1999)). In fact, removing calcium ions from solution using EGTA causes increased deamidation and aggregation. However, this effect was observed only with Ca ²⁺ ions; Other divalent cations Mg ²⁺ , Mn ²⁺ and Zn ²⁺ have been shown to cause destabilization of rhDNase. Similar effects were observed with factor VIII. Ca ²⁺ and Sr ²⁺ ions stabilized

- 61 045553 protein, while others, such as Mg ²⁺ , Mn ²⁺ and Zn ²⁺ , Cu ²⁺ , and Fe ²⁺ , destabilized the enzyme (Fatouros, A., et al., Int. J. Pharm., 155, 121-131 (1997).In a separate study of factor VIII, a significant increase in the rate of aggregation was observed in the presence of Al ³⁺ ions (Derrick TS, et al., /. Pharm. Sci., 93(10): 2549- 57 (2004). The authors note that other excipients, such as buffer salts, are often contaminated with Al ³⁺ ions, and illustrate the need to use appropriate quality excipients in finished products.

Other common excipient components: pharmaceutical preservatives.

Preservatives are necessary when developing multidose parenteral products that involve more than one extraction from a single container. Their main function is to inhibit the growth of microbes and ensure the sterility of the product throughout the shelf life or period of use of the medicinal product. 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 that include preservatives can be challenging. Preservatives almost always have a destabilizing effect (aggregation) on proteins, and this has become a major factor limiting their use in multidose protein formulations (Roy S, et al., J Pharm ScL, 94(2): 382-96 (2005)).

To date, most protein preparations have been developed for one-time use only. However, when multi-dose formulations are possible, they have the added benefit of patient convenience and increased marketability. A good example is human growth hormone (hGH), where the development of canned formulations has led to the commercialization of more convenient, multi-purpose injection pens. There are at least four such pen devices containing canned hGH formulations currently available on the market. Norditropin® (liquid, Novo Nordisk), Nutropin AQ® (liquid, Genentech) & Genotropin (lyophilized dual chamber cartridge, Pharmacia & Upjohn) contain phenol, while Somatrope® (Eli Lilly) contains m-cresol.

When developing formulations for canned dosage forms, several aspects must be taken into account. The effective concentration of the preservative in the medicinal product must be optimized. This requires testing a given preservative in dosage form at concentration ranges that provide antimicrobial effectiveness without compromising protein stability. For example, three preservatives were successfully tested in the development of a liquid formulation for interleukin-1 receptor (type I) using differential scanning calorimetry (DSC). Preservatives have been ranked according to their effect on stability at concentrations commonly used in marketed products (Remmele RL Jr., et al., Pharm Res., 15(2): 200-8 (1998)).

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

Some preservatives may cause injection site reactions, which is another factor to consider when choosing a preservative. In clinical trials that focused on evaluating the preservatives and buffers in Norditropin, pain perception was less in formulations containing phenol and benzyl alcohol compared to a formulation containing m-cresol (Kappelgaard A.M., Horm Res. 62 Suppl 3:98- 103 (2004)). Interestingly, among the commonly used preservatives, benzyl alcohol has anesthetic properties (Minogue SC, and Sun DA., AnesthAnalg., 100(3): 683-6 (2005)). In various aspects, the use of preservatives provides benefits that outweigh any side effects.

Methods for preparing pharmaceutical compositions.

The present invention also provides methods for preparing pharmaceutical compositions.

These methods further include one or more of the following steps: adding a stabilizing agent, as described herein, to the mixture before lyophilization, adding at least one agent selected from a bulking agent, an osmolality adjusting agent, and a surfactant, each of which, as described herein, to the specified mixture before lyophilization.

Standard practice for reconstituting lyophilized material is to add a volume of pure water or sterile water for injection (WFI) (usually equivalent to the volume removed during lyophilization), although dilute solutions of antibacterial agents are sometimes used in the manufacture of parenteral pharmaceuticals (Chen, Drug Development and Industrial Pharmacy, 18:1311-1354 (1992)). Accordingly, methods for preparing reconstituted rVWF compositions are provided, including the step of adding a diluent to the lyophilized rVWF composition of the invention.

Lyophilized material can be reconstituted as an aqueous solution. A variety of aqueous vehicles, for example, sterile water for injection, preservative water for repeated use, or water with appropriate amounts of surfactants (for example, an aqueous suspension that contains the active compound in admixture with excipients suitable for the production of aqueous suspensions). In various aspects, such excipients are suspending agents, such as, but not limited to, sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and acacia gum; the dispersing or wetting agent is a naturally occurring phosphatide, such as, but not limited to, lecithin, or alkylene oxide fatty acid condensates, such as, but not limited to, polyoxyethylene stearate or ethylene oxide condensates with long chain aliphatic alcohols, such as, but not limited to, heptadecaethyleneoxycetanol or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol, such as polyoxyethylene sorbitan monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example, and without limitation, polyethylene sorbitan monooleate. In various aspects, aqueous suspensions also contain one or more preservatives, such as, but not limited to, ethyl or n-propyl phydroxybenzoate.

Illustrative formulation of mat-rVWF for administration.

In some embodiments, these methods provide an improved formulation that produces a final product with high potency (high matrVWF concentration and increased long-term stability) to reduce treatment volume (100 to 10,000 IU/mL). In some embodiments, the concentration of mat-rVWF in the administration composition is from about 100 to 10,000 IU/ml. In some embodiments, the concentration of mat-rVWF in the administration composition is from about 500 to 10,000 IU/ml. In some embodiments, the concentration of mat-rVWF in the administration composition is from about 1000 to 10,000 IU/ml. In some embodiments, the concentration of mat-rVWF in the administration composition is from about 2000 to 10,000 IU/ml. In some embodiments, the concentration of mat-rVWF in the administration composition is from about 3,000 to 10,000 IU/ml. In some embodiments, the concentration of matrVWF in the administration composition is from about 4,000 to 10,000 IU/ml. In some embodiments, the concentration of mat-rVWF in the administration composition is from about 5,000 to 10,000 IU/ml. In some embodiments, the concentration of mat-rVWF in the administration composition is from about 6,000 to 10,000 IU/ml. In some embodiments, the concentration of mat-rVWF in the administration composition is from about 7,000 to 10,000 IU/ml. In some embodiments, the concentration of mat-rVWF in the administration composition is from about 8,000 to 10,000 IU/ml. In some embodiments, the concentration of mat-rVWF in the administration composition is from about 9,000 to 10,000 IU/ml. In some embodiments, mat-rVWF is co-formulated with recombinant factor VIII (rFVIII). In some embodiments, rFVIII is full-length FVIII. In some embodiments, rFVIII is full-length and chemically modified. In some embodiments, rFVIII includes a FVIII fusion protein containing a FIX activation peptide in place of the B domain. In some embodiments, rFVIII is a fusion of FVIII containing a truncated glycosylation B domain. In some embodiments, FVIII is a variant with the FVIII B domain deleted. In some embodiments, FVIII is a chemically modified variant of the FVIII B domain deleted variant. In some embodiments, a co-formulation of mat-rVWF with rFVIII is prepared prior to the freeze-drying or completion of filling step and stored by mixing the components in vitro or in a column procedure (eg, adding FVIII during the purification method).

In some embodiments, the administration composition contains one or more zwitterionic compounds, including, for example, amino acids such as histidine, glycine, arginine. In some embodiments, the administration composition includes a component with amphipathic characteristics having at least one hydrophobic and one hydrophilic group, including, for example, polysorbate 80, octyl pyranoside, dipeptides and/or amphipathic peptides. In some embodiments, the administration composition contains a non-reducing sugar or sugar alcohol or disaccharides, including, for example, sorbitol, mannitol, sucrose or trehalose. In some embodiments, the administration composition includes a non-toxic water-soluble salt, including, for example, sodium chloride, which results in physiological osmolality. In some embodiments, the administration composition has a pH in the range of 6.0 to 8.0. In some embodiments, the administration composition has a pH of about 6.0, about 6.5, about 7, about 7.5, or about 8.0. In some embodiments, the administration composition contains one or more divalent cations that stabilize rVWF, including, for example, Ca ²⁺ , Mg ²⁺ , Zn ²⁺ , Mn ²⁺ and/or combinations thereof. In some embodiments, the administration composition contains from about 1 mM to about 50 mM glycine, from about 1 mM to about 50 mM histidine, from about zero 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 with a pH of about 7.0 and physiological osmolarity at the time of administration .

In some embodiments, the administration composition may be freeze-dried. In some embodiments, the administration composition is stable and can be stored in a liquid state at a temperature of from about 2°C to about 8°C, and from about 18°C to about 25°C. In some embodiments, the administration composition is stable and can be stored in a liquid state at a temperature of from about 2°C to about 8°C. In some embodiments, the administration composition is stable and can be stored in a liquid state at a temperature of from about 18°C to about 25°C.

Purpose.

For administration of the compositions to humans or experimental animals, in one aspect, the compositions contain one or more pharmaceutically acceptable carriers. The phrases pharmaceutically or pharmacologically acceptable refer to molecular entities and compositions that are stable, inhibit protein degradation such as aggregation and degradation products, and, in addition, do not cause allergic or other adverse reactions when administered using methods well known in the art techniques as described below. Pharmaceutically acceptable carriers include any and all clinically useful solvents, dispersion media, coatings, antibacterial and fungicidal agents, isotonic agents and absorption retarding agents, and the like, including the agents described above.

Pharmaceutical formulations are administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or by intracranial injection. As used herein, the term parenteral includes subcutaneous injection, intravenous, intramuscular, intracisternal injection or infusion methods. Administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and/or surgical implantation at a specific site is also considered. Typically, the compositions contain virtually no pyrogens or other impurities that may be harmful to the recipient.

Single or multiple administration of the compositions is carried out at the dose level and nature of the doses that are selected by the attending physician. For the prevention or treatment of a disease, the appropriate dosage depends on the type of disease being treated as defined above, the severity and course of the disease, whether the drug is being administered for prophylactic or therapeutic purposes, previous therapy, the patient's medical history and response to the drug, and at the discretion of the attending physician.

Sets.

As a further aspect, the invention includes kits that contain one or more lyophilized compositions packaged in such a way as to facilitate their use for administration to subjects. In one embodiment, such a kit contains a pharmaceutical composition described herein (e.g., a composition containing a therapeutic protein or peptide) packaged in a container, such as a sealed bottle or container, with a label affixed to the container or included in packaging that describes use of the compound or composition when applying the method. In one embodiment, the pharmaceutical composition is packaged in a container such that the amount of free space in the container (eg, the amount of air between the liquid composition and the top of the container) is very small. It is preferable that the amount of free space be negligible (eg, almost zero). In one embodiment, the kit contains a first container containing a therapeutic protein or peptide composition and a second container containing a physiologically acceptable solution for reconstituting the composition. In one aspect, the pharmaceutical composition is packaged in a unit dosage form. The kit may further comprise a device suitable for administering the pharmaceutical composition according to a particular route of administration. Preferably, the kit contains a label that describes the use of the pharmaceutical compositions.

Doses.

The dosage regimen included in the treatment of a condition described herein will be determined by the attending physician, taking into account various factors that modify the effect of drugs, such as the patient's age, condition, body weight, sex and diet, severity of infection, timing of administration, and other clinical factors. factors. For example, a typical dose of recombinant VWF according to this invention is approximately 50 U/kg, which is equal to 500 μg/kg.

In one aspect, the compositions of this invention are administered by an initial bolus followed by a continuous infusion to maintain therapeutic levels of the drug product in the blood. As another example, a compound of the invention is 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 the individual patient. Dosing frequency depends on the pharmacokinetic parameters of the agents and the route of administration. The optimal pharmaceutical composition is determined by one skilled in the art depending on the route of administration and the desired dosage. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pp. 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 agents. Depending on the route of administration, the appropriate dose is calculated according to body weight, body surface area or organ size. Suitable doses can be established using conventional blood dose assays in combination with appropriate dose-response data. The final dosage regimen is determined by the attending physician, taking into account various factors that modify the effect of the drugs, such as the specific activity of the drug, the severity of the injury and the patient's response, the patient's age, condition, weight, sex and diet, severity of infection, time of administration and other clinical factors . As research progresses, more information will become available regarding appropriate dosage levels and duration of treatment for various diseases and conditions.

Examples.

The following non-limiting examples are presented for illustrative purposes only to facilitate a more complete understanding of typical embodiments.

These examples should not be construed as limiting any of the embodiments described in this invention, including those relating to methods of treating acquired and genetic von Willebrand disease.

Example 1: Purification of mature rVWF using a cation exchanger to separate cVWF propeptide from mature rVWF.

Example 1 represents the purification of mature rVWF on a cation exchanger (cation exchange resin (CEX)). The rVWF propeptide (rVWF-PP) remained associated with rVWF after furin maturation and was dissociated with sodium citrate as a chelating agent at neutral pH before application to the CEX resin. Most of the rVWF propeptide passed through the cation exchange resin. And the remaining rVWF propeptide was removed after the washing step. Sodium citrate was used as a component of a buffer substance and as a chelating agent.

At the industrial level, VWF, particularly recombinant VWF (rVWF), is synthesized and expressed together with rFVIII in a genetically engineered CHO cell line. The function of coexpressed rVWF is to stabilize rFVIII during cell culture. rVWF is synthesized in the cell as a pro form containing a large propeptide attached to the N-terminus. After maturation in the endoplasmic reticulum and Golgi apparatus, rVWF-PP is cleaved by the cellular protease furin and secreted as a homopolymer of identical subunits, consisting of dimers of the expressed protein. However, maturation is incomplete, resulting in a product containing a mixture of rVWF-PP and mature VWF.

After the monoclonal antibody capture step for recombinant factor VIII, a flow buffer containing rVWF (also called monoclonal antibody effluent) is loaded onto an anion exchanger (anion exchange resin (AEX)). rVWF binds to the anion exchanger and matures with furin in the presence of calcium. RVWF was eluted from the anion exchanger with increasing conductivity. The product containing the eluate was treated by diluting 1:2 with 60 mM sodium citrate, pH 7.6, to a conductivity of 13.39 mS/cm and pH 7.39. The treated aqueous solution was loaded into a UNOsphere™ S Cation Exchange Media column (Bio Rad, cat. no: 156-0115) with an internal diameter of 15 mm, a bed height of 14.0 cm and a volume of 24.74 ml with a flow rate of 100 cm/h , and then washed with 5 OK (column volumes) 10 mM NaCl, 30 mM Na citrate, pH 7.6 ± 0.2 to remove host cell proteins (HCP) and rVWF-PP. rVWF was eluted by increasing conductivity using a linear gradient at a flow rate of 60 cm/h in 6 OK from 10 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 to a buffer of 500 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2. The main peak of the eluate was divided into two parts to separate low molecular weight rVWF multimers and high molecular weight rVWF multimers.

In fig. Figure 1 shows the purification of mature rVWF on a cation exchanger presented in Example 1.

In fig. 2 shows a table of cleaning results.

In fig. 3 shows a silver-stained protein gel and Western blot illustrating the separation of rVWF and rVWF-PP by the method described in Example 1.

Examples 2 and 3: Optimized method described in Example 1 for commercial production of rVWF.

Examples 2 and 3 demonstrate an optimized method described in Example 1 for commercial production of rVWF.

For Examples 2 and 3, a pilot plant was created for the fermentation of rVWF and rFVIII. The method was used for a simplified purification method used to obtain high rVWF

- 65 045553 of a certain degree of purity for biochemical characterization.

The capture step was performed using tandem chromatography, which combined affinity chromatography and anion exchange chromatography in one process step. rFVIII was bound to the FVIII-mAb column at a temperature of 2-8°C based on the immunoaffinity chromatography method. At this stage, rFVIII can be separated from rVWF. The running buffer containing rVWF was diluted in the same chromatographic system with purified water and loaded directly onto the AEX column. Maturation of recombinant furin on an AEX column was carried out after increasing the temperature from +15 to 28°C. Furin-ripened rVWF was eluted by stepwise elution by increasing conductivity. A post-purification stage was also carried out.

The AEX eluate containing rVWF was diluted with 10 mM Na-citrate buffer, pH 7.6 and applied to a UNOsphere™ S Cation Exchange Media column (Bio Rad, cat. no: 156-0115), having an internal diameter of 15 mm, bed height 14.0 cm and column volume 25 ± 0.5 ml at a flow rate of 100 cm/h. After a wash step with 10 mM NaCl, 30 mM Na citrate, 2 mM citric acid pH 7.6 ± 0.2, rVWF was eluted with increasing conductivity using a linear gradient at a flow rate of 65 cm/h in 6 OA from 10 mM NaCl, 30 mm. Sodium citrate, pH 7.6 ± 0.2, to a buffer containing 500 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2. The main peak of the eluate was collected as eluate (pooled eluate) for analytical purposes.

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

In fig. Figure 4 shows a block diagram of the experimental setup for Examples 2 and 3.

In fig. Figure 5 shows the chromatogram for example 2 and the chromatography scheme used for examples 2 and 3.

In fig. Figure 6 shows a table of the reagents used and a table of results for example 2.

In fig. Figure 7 shows another chromatogram from example 2 and a table of results for example 3.

In fig. 8 shows a silver-stained protein gel illustrating the separation of rVWF and rVWF propeptide by the method of Example 2 and Example 3.

In fig. 9 is a Western blot illustrating the separation of rVWF and rVWF propeptide by the method of Example 2 and Example 3.

Example 4 Method for industrial production of rVWF by separating rVWF and rVWF-PP using size exclusion chromatography.

Example 4 is an optimized method for the commercial production of rVWF by separating rVWF and rVWF propeptide (rVWF-PP) using size exclusion chromatography. Sodium citrate is added to the SEC running buffer to ensure efficient separation of rVWF and rVWF-PP.

RVWF containing ultrafiltered UNOsphere™ S-eluate was loaded directly into a row of two sequential SEC Superose 6 preparation columns (GE Healthcare, cat. no: 289913-16), each with an internal diameter of 16 mm bed height of 82.0 cm (2x41 cm), and the volume of both columns was approximately 165 ml. The loading buffer was applied at a speed of 7 cm/h. The working buffer was 20 mM HEPES free acid, 150 mM NaCl, 15 mM sodium citrate dihydrate, pH 7.5 ± 0.2. Size exclusion chromatography was carried out under isocratic conditions at a linear flow rate of 12 cm/h.

In fig. 10 shows the chromatogram, chromatography scheme and buffer compositions for Example 4.

In fig. Figure 11 shows a table of results for example 4.

In fig. 12 shows a silver-stained protein gel and Western blot illustrating the separation of rVWF and rVWF propeptide by the method of Example 4.

Example 5: Optimized method for commercial production of mature rVWF by separating rVWF and rVWF-PP using size exclusion chromatography.

Example 5 presents a method for separating rVWF and rVWF-PP by size exclusion chromatography using pH-altered rVWF containing starting material on size exclusion chromatography.

RVWF containing ultrafiltered UNOsphere™ S eluate was adjusted to pH 7.5 ± 0.2 with 1 M glycine pH 9.0 before loading onto the column. This solution was loaded into a row of two sequential SEC Superose 6 preparation columns (GE Healthcare, cat. no: 28-9913-16), each with an internal diameter of 16 mm, a layer height of 82.0 cm (2x41 cm), and a volume of both column was approximately 165 ml. The loading buffer was used at a flow rate of 7 cm/h. The working buffer contains 20 mM HEPES free acid and 150 mM NaCl, pH 7.5 ± 0.2. Size exclusion chromatography was carried out under isocratic conditions at a linear flow rate of 12 cm/h.

In fig. 13 shows the chromatogram, chromatography scheme and buffer compositions for Example 5.

In fig. Figure 14 shows a table of results for example 5.

Example 6. CEX method for purification of rVWF from rVWF propeptide without adding chelating agents on UNOsphere™ S.

Example 6 is a CEX method without the addition of chelating agents on ultrafiltrated UNOsphere™ S. This method is representative of a known method for purifying mature rVWF from rVWF propeptide. The method does not use a buffer containing a chelating agent and/or a buffer having a pH of 7.0 or higher.

After the monoclonal antibody step to capture recombinant factor VIII, the flow buffer containing rVWF is loaded onto the anion exchanger. rVWF binds to the anion exchanger and matures with furin in the presence of calcium. RVWF was eluted from the anion exchanger with increasing conductivity. The product containing the eluate was then loaded onto a UNOsphere™ S Cation Exchange Media column (Bio Rad, Part No: 156-0115) with a 15 mm ID, 14.2 cm bed height and 25.09 mL volume at a flow rate of 100 cm/h followed by washing with 10 OK 10 mM Tris-HCl, 100 mM Na acetate, 85 mM NaCl, pH 6.5 ± 0.2 to remove NCP and rVWF propeptide. rVWF was eluted in one step using 100 mM Na acetate, 500 mM NaCl, 100 mM glycine, 3 mM CaCl ₂ , pH 7.5 ± 0.2 at a flow rate of 65 cm/h. The main peak of the eluate was collected as the product containing fraction.

In fig. 15 shows the chromatogram, chromatography scheme and buffer compositions, as well as conditions for Example 6.

In fig. Figure 16 shows a table of results for example 6.

Example 7: SEC method for purifying rVWF from rVWF propeptide without prior addition of chelating agents or increased pH.

Example 7 represents the SEC method without prior addition of chelating agents or elevated pH. This method is representative of a known method for purifying mature rVWF from rVWF propeptide. The SEC method does not include a buffer containing a chelating agent and/or a buffer having a pH of 7.0 or greater that is used to modify the starting fraction (material) containing rVWF and the residual rVWF propeptide.

Recombinant RVWF containing ultrafiltered UNOsphere™ S-eluate was loaded directly into an array of two sequential SEC Superose 6 preparation columns (GE Healthcare, Cat. No: 28-9913-16), each with an internal diameter of 16 mm bed height of 82.0 cm (2x41 cm), and the volume of both columns was approximately 165 ml. The loading buffer was used at a flow rate of 7 cm/h. The working buffer contained 20 mM HEPES free acid, 150 mM NaCl, pH 7.5 ± 0.2. Size exclusion chromatography was carried out under isocratic conditions at a linear flow rate of 12 cm/h.

In fig. 17 shows the chromatogram, chromatography scheme and buffer compositions for Example 7.

In fig. Figure 18 shows a table of results for example 7.

Example 8 Separation of rVWF from rVWF propeptide using anion exchange chromatography and cation exchange chromatography.

Example 8 presents the purification of mature rVWF on a cation exchanger. The starting material was obtained from the current rVWF manufacturing process after the AEX Mustang Q step. RVWF containing the run-through buffer from the AEX Mustang Q step was treated with SD/VI and diluted with a chelating agent-containing buffer to dissociate the rVWF/rVWF-propeptide complex. The diluted material was applied to CEX resin (Unosphere S). Most rVWF-PP, host cell proteins (HCPs), and low molecular weight rVWF multimers pass through the cation exchange resin. The remaining rVWF-PP was removed after the washing step. Bound high molecular weight rVWF multimers were subsequently eluted by sodium ion-induced increased conductance.

At the industrial level, VWF, particularly recombinant VWF (rVWF), is synthesized and expressed together with rFVIII in a genetically engineered CHO cell line. The function of coexpressed rVWF is to stabilize rFVIII during cell culture. rVWF is synthesized in the cell as a pro form containing a large propeptide attached to the N-terminus. After maturation in the endoplasmic reticulum and Golgi apparatus, the propeptide is cleaved off by the cellular protease furin and secreted as a homopolymer of identical subunits, consisting of dimers of the expressed protein.

However, maturation is incomplete, resulting in a product containing a mixture of propeptide and mature VWF.

Following the monoclonal antibody step to capture recombinant factor VIII, the flow buffer containing rVWF is loaded onto a Fractogel TMAE anion exchanger. rVWF binds to the anion exchanger and matures with furin in the presence of calcium. RVWF was 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 feed material for the CEX step is the effluent from the Mustang Q (MUQ) filtration step, which is treated with solvent and detergents to inactivate lipid enveloped viruses. To inactivate viruses, MUQ wastewater is incubated with a mixture of two detergents, such as Triton-X100 (1%) and polysorbate 80 (0.3%), and the organic solvent tri-n-butyl phosphate (0.3%) for one hour at room temperature. The product containing MUQ_flow buffer was treated by diluting 1:2 with 60 mM sodium citrate, pH 7.6, to a conductivity of 21.9 mS/cm and pH 7.16. High

- 67 045553 conductivity was selected to ensure removal of rVWF propeptide (rVWF-PP) and low molecular weight rVWF multimers to utilize the resin's ability for the desired high molecular weight rVWF multimers. The treated dilution buffer was loaded into a UNOsphere™ S Cation Exchange Media column (Bio Rad, cat. no: 156-0115) with an internal diameter of 10 mm, a bed height of 14.3 cm and a volume of 11.23 ml with a flow rate of 100 cm/ h. After loading, a first wash (re-equilibration) was performed using 5 OC 10 mM NaCl, 30 mM sodium citrate, pH 7.6 ± 0.2 to remove weakly bound HCP and rVWF propeptide.

The second wash to remove tightly bound NCP and rVWF propeptide was carried out in 40% increments with 500 mM NaCl, 30 mM Na citrate, pH 7.6 ± 0.2 in 10 mM NaCl, 30 mM Na citrate, pH 7.6 ± 0, 2 (wash 2).

Elution was carried out in two phases: (1) the first phase included a step of 45% 500 mM NaCl, 30 mM Na citrate, pH 7.6 ± 0.2 in 10 mM NaCl, 30 mM Na citrate, pH 7.6 ± 0.2 (eluate 1 or E1), a (2) the second phase 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 Na citrate, pH 7 .6 ± 0.2. (eluate 2 or E2) in 6 column volumes. Wash 2 to the end of the gradient was performed at a flow rate of 65 cm/h.

In fig. 19 shows the chromatogram, chromatography scheme and buffer compositions for Example 8.

In fig. Figure 20 shows a table of results for Example 8.

In fig. 21 is a silver-stained protein gel illustrating the separation of rVWF and rVWF propeptide by the method of Example 8.

In fig. 22 is a Western blot illustrating the separation of rVWF and rVWF propeptide by the method of Example 8. The 1% agarose gel demonstrates the multimeric structure of the products.

In fig. 23 is a Western blot illustrating the separation of rVWF and rVWF propeptide by the method of Example 8.

Example 9 Separation of rVWF from rVWF propeptide using anion exchange chromatography and cation exchange chromatography.

Example 9 presents an optimized purification of mature rVWF on a cation exchanger. The starting material was obtained from the current r-VWF production process after the Mustang Q AEX step. The RVWF containing the run-through buffer from the Mustang Q AEX step was treated with SD/VI and diluted with a chelating agent-containing buffer to dissociate the rVWF/rVWFpropeptide complex. The diluted material was applied to CEX resin (Unosphere S). Most rVWF-PP, host cell proteins, and low molecular weight rVWF multimers pass through the cation exchange resin. The remaining rVWF-PP was removed after the washing step. Bound high molecular weight rVWF multimers were subsequently eluted by a gradient by increasing sodium ion-induced conductance.

At the industrial level, VWF, particularly recombinant VWF (rVWF), is synthesized and expressed together with rFVIII in a genetically engineered CHO cell line. The function of coexpressed rVWF is to stabilize rFVIII during cell culture. rVWF is synthesized in the cell as a pro form containing a large propeptide attached to the N-terminus. After maturation in the endoplasmic reticulum and Golgi apparatus, the propeptide is cleaved off by the cellular protease furin and secreted as a homopolymer of identical subunits, consisting of dimers of the expressed protein. However, maturation is incomplete, resulting in a product containing a mixture of propeptide and mature VWF.

Following the monoclonal antibody step to capture recombinant factor VIII, the flow buffer containing rVWF is loaded onto a Fractogel TMAE anion exchanger. r-VWF is on the anion exchanger and matures with furin in the presence of calcium. RVWF was 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 feed material for the CEX step is the effluent from the Mustang Q (MUQ) filtration step, which is treated with solvent and detergents to inactivate lipid enveloped viruses.

To inactivate viruses, MUQ wastewater is incubated with a mixture of two detergents Triton-X100 (1%) and polysorbate 80 (0.3%) and the organic solvent tri-n-butyl phosphate (0.3%) for one hour at room temperature. The product containing MUQ_flow buffer was treated by diluting 1:2 with 60 mM sodium citrate, pH 7.6, to a conductivity of 21.9 mS/cm and pH 7.16. High conductivity is selected to ensure removal of rVWF-propeptide and low molecular weight rVWF to utilize the resin's capacity for the desired high molecular weight r-VWF. The treated dilution buffer was loaded into a UNOsphere™ S Cation Exchange Media column (Bio Rad, cat. no: 156-0115) with an internal diameter of 10 mm, a bed height of 14.3 cm and a volume of 11.23 ml with a flow rate of 100 cm/ h followed by a first wash (re-equilibration) of 5 OK 10 mM NaCl, 30 mM Na citrate, pH 7.6 ± 0.2 to remove loosely bound HCP and rVWF propeptide.

The second wash (wash 2) to remove tightly bound NCP and rVWF-propeptide was carried out in 36% increments with 500 mM NaCl, 30 mM Na citrate, pH 7.6 ± 0.2 in 10 mM NaCl, 30 mM Na citrate , pH 7.6 ± 0.2 in 5 column volumes.

Elution was carried out with a gradient from 36% 500 mM NaCl, 30 mM Na citrate, pH 7.6 ± 0.2 in 10 mM NaCl, 30 mM Na citrate, pH 7.6 ± 0.2 to 100% 500 mM NaCl, 30 mM Na 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 representing the desired product contains a pool of fractions ranging from >50% 500 mM NaCl, 30 mM Na Citrate, pH 7.6 ± 0.2 in 10 mM NaCl, 30 mM Na Citrate, pH 7.6 ± 0, 2 to 76% of 500 mM NaCl, 30 mM Na citrate, pH 7.6 ± 0.2 in 10 mM NaCl, 30 mM Na citrate, pH 7.6 ± 0.2. Washing and elution were performed at a flow rate of 50 cm/h.

In fig. Figure 24 shows the chromatogram, chromatography scheme, and buffer compositions for Example 9.

In fig. Figure 25 shows a table of results for example 9.

In fig. Figure 26 shows the product table for example 9.

In fig. 27 is a silver-stained protein gel illustrating the separation of rVWF and rVWF propeptide by the method of Example 9.

In fig. 28 shows a Western blot illustrating the separation of rVWF and rVWF propeptide by the method of Example 9. The 1% agarose gel demonstrates the multimeric structure of the products.

In fig. 29 is a Western blot illustrating the separation of rVWF and rVWF propeptide by the method of Example 9.

Purification steps of rVWF in the presence of chelating agents and/or increased pH showed high rates of removal of r-VWF propeptide and host cell proteins. Removal of the r-VWF propeptide on the cation exchanger is based on the fact that rVWF-PP does not bind to the cation exchanger under conditions of the presence of chelating agents and/or increased pH. Removal of rVWF propeptide by size exclusion chromatography is based on the fact that size separation is effective in the presence of chelating agents and/or increased pH.

In fig. 30 shows the purity of the product containing fractions obtained from the enhanced cation exchange chromatography (CEX) used in Examples 1, 2, 3, 6, 8 and 9.

In fig. 31 shows the removal rate of product-related impurities for Examples 1, 2, 3, 6, 8 and 9.

In fig. 32 shows the purity of the product containing the enhanced size exclusion chromatography (SEC) fractions used in Examples 4 and 5.

In fig. 33 shows the removal rate of product-related impurities for Examples 4 and 5.

Links

US Patent No. 8058411; Method for producing mature VWF from VWF pro-peptide. Inventors: Wolfgang Mundt, Artur Mitterer, Meinhard Hasslacher, Christa Mayer.

US Patent No. 6465624; Purification of von Willebrand factor by cation exchange chromatography. Inventors: Bernhard Fischer, Oyvind L. Schonberger, Artur Mitterer, Christian Fiedler, Friedrich Dorner, Johann Eibl.

Example 10 Separation of rVWF from rVWF propeptide using anion exchange chromatography.

This study illustrates the dissociation of furin-treated mature VWF/VWF-PP complex into mature VWF and VWF-PP using anion exchange chromatography and an elution buffer with elevated pH (eg, pH 8.5) containing a chelating agent (EDTA). The separation was carried out on an anion exchanger (AEX), in particular on Fractogel TMAE 650 (M). Solvent-detergent treatment to inactivate viruses was also carried out on the column for about 1 hour. Details of the chromatographic experiment are presented in FIG. 34-36.

In fig. 34 shows the buffer compositions and materials used in the TMAE separation method.

In fig. 35 shows loading conditions for the mature VWF/VWF-propeptide complex treated with furin.

In fig. 36 shows details of the buffers, conditions, parameters and flow rates of the chromatographic method.

In fig. 37 shows a chromatogram of the dissociation of furin-treated mature VWF/VWF-propeptide complex into mature VWF and VWF-propeptide (VWF-PP). It shows the removal of VWF-PP from the fraction containing mature VWF.

In fig. 38 shows another separation chromatogram of mature VWF and VWF-propeptide (VWF-PP). It shows the removal of VWF-PP from the fraction containing mature VWF.

Example 11: Improvements in various chromatographic techniques for the separation of mature VWF (matVWF) and VWF propeptide (VWF-PP).

The first study compared two methods for purifying recombinant mature VWF.

In fig. 39 shows a diagram of two methods for isolating mature VWF. In one method, post-processing steps such as the mAb effluent (MABEffl) step, the TMAE anion exchange chromatography capture and maturation on column (TMC) step, and the Mustang Q negative anion exchange chromatography (MUQ) step include solvent-detergent treatment

- 69 045553 (SDT) for virus inactivation, cation exchange chromatography (CAT), ultrafiltration concentration (UFA), size exclusion chromatography (SEC) and dialysis ultrafiltration concentration (DUF) to obtain bulk drug substance (mature VWF). In another method, subsequent processing steps include an enhanced cation exchange chromatography (CAT) step followed by a dialysis-ultrafiltration (DUF) concentration step to obtain bulk drug substance, and do not include SEC.

In fig. 40 is a table highlighting some of the benefits of the advanced cation exchange chromatography technique (CAT 2.0) described herein and shown in FIG. 39. The advanced CAT method can remove: host cell contaminants with a reduction factor greater than 1000, VWF-PP with a reduction factor greater than 2000, and residual FVIII with a reduction factor less than 10. The CAT method can be used to separate and combine VWF multimers. Additionally, this method can replace size exclusion chromatography as a post-purification step to isolate the active VWF fraction and remove remaining host cell-derived impurities and VWF-PP.

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

In fig. 41 is a diagram of two chromatograms showing the separation of r-VWF propeptide using size exclusion chromatography with standard SEC buffer (SQA buffer) or modified SEC buffer (SQC buffer). In fig. Figure 42 is a table that shows some of the benefits of using the SQC buffer. For example, a method using SQC buffer can remove host cell impurities with a reduction factor greater than 100 and residual FVIII with a reduction factor less than 10. Surprisingly, it can remove VWF-PP such that impurity levels are less than 2 μg/1000 units .

Thus, this example describes methods for improving the separation of mature VWF from VWF-PP.

Example 12: Development of an improved CAT stage (UNO_S).

The downstream 1st generation recombinant von Willebrand factor (rVWF) process, starting with a monoclonal antibody (MAB) stream, includes a post-purification 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 the downstream process. The high molecular weight subfraction of rVWF is the bulk drug substance (BDS) that is finally prepared to form the final drug product (FDP).

For the 2nd generation rVWF downstream process, it has been proposed to replace the SEC step with an improved cation exchange chromatography method and to separate high, low and molecular weight rVWF multimers as well as rVWF propeptides using an alternative cation exchange (CAT) elution procedure (gradient elution instead of step elution) . This example outlines experiments in the CAT purification stage of the 2nd generation rVWF post-treatment. New process parameters such as pH and conductivity of the CAT load, conductivity and duration of column washing steps, and eluate pooling criteria were investigated on a small scale to obtain a scalable and robust downstream process step.

1. Task.

The 1st generation rVWF downstream process (VONVENDI®) begins with a capture step on TMAE-Sepharose (TMS step) using an ADVATE® MAB stream as the feed solution, followed by a Mustang Q filtration step to remove host cell CHO DNA. A solvent/detergent (S/D) step was then performed to inactivate potential lipid enveloped viruses, followed by a weak cation exchanger UNO_Sphere S (UNO_S) resin post-purification step (CAT step). The CAT step is designed to remove the S/D reagents 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 the downstream process. The high molecular weight subfraction of rVWF is BDS, which is finally formulated to produce FDP.

For the subsequent 2nd generation rVWF process, it was proposed to eliminate the SEC step and replace it with an improved cation exchange chromatography method.

In a series of five experiments, separation of high and low molecular weight rVWF multimers as well as removal of rVWF propeptides was achieved using a CAT gradient elution procedure. The new gradient elution mode was able to replace the stepwise elution procedure used in the subsequent 1st generation process. In this example, details

- 70 045553 five experiments are described for the CAT purification stage of the rVWF 2nd generation tertiary treatment. All experiments were performed according to the study design described herein.

2. Introduction and state of the art.

The current report describes the development of the 2nd generation (Gen 2) T process by combining the two subsequent stages of VWF block operation CAT and SEC, which are currently used in the 1st generation (Gen 1) procedure. In a series of experiments, process parameters that were identified in the risk assessment and considered important for performing the chromatographic step of the CAT were investigated. This study was based on a scaled-down model of the current rVWF production process. This process was established at Orta for the production of clinical phase III material and scaled up to industrial scale (MFG) for commercial production (Fig. 43A). To make it easier to understand the changes made to the CAT block work phase described in this report, below is a summary of the process of the Gen 1 rVWF downstream block work stages S/D, CAT, and SEC.

Used in the Gen 1 process, the rVWF CAT post-purification step is a chromatographic cation exchange process on UNO_Sphere S, an acrylamide-based macroporous media with a strong sulfonic cation exchange ligand. The feed material for the post-treatment stage is the effluent from the MUQ anion exchange filtration stage, which is treated with solvent and detergents to inactivate lipid-enveloped viruses. To inactivate viruses, MUQ wastewater is incubated 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%) for one hour at room temperature. temperature. Before processing, the product solution is filtered through a 0.2 µm membrane filter to remove any potentially present particles. After virus inactivation, the product solution is diluted with approximately one volume of water to reduce the concentration of S/D reagents, adjusting the conductivity for the loading step on the CAT column. PH is not regulated. The primary purpose of CAT chromatography is to remove S/D reagents and further reduce process-related impurities, including media components such as soy peptone, and other impurities such as rfurin (recombinant furin), rFVIII polypeptides and proteins and DNA, received from SNO. After the CAT installation stage, the resulting product fraction (CAT-E) is further processed by size exclusion chromatography (SEC) on Superose 6 resin. The loading material for the SEC post-purification stage is the CAT eluate pool at the cation exchange post-purification stage on UNO_Sphere S. Since the loading volume for the SEC column is limited To achieve reasonable resolution, the CAT eluate pool is concentrated approximately 15-fold by ultrafiltration using a cellulose-based membrane cassette with a 30°kDa cutoff (UFA step). In clinical phase III production scale, the ultrafiltration concentration (UFA) concentrate is separated into two fractions, which are processed separately on a SEC column. This measure was implemented to keep SEC column volume and column diameter low. The buffer matrix, as well as the conductivity and pH of 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 the SEC step is to ultimately remove contaminants from CHO host cell proteins and serves as the primary step for removing the product-bound contaminant rVWF propeptide generated during the initial capture step on TMAE-Sepharose (TMS step). Additionally, the SEC step separates rVWF multimers based on their size, allowing the pooling scheme to be used to enrich high molecular weight rVWF multimers that contribute to the product's ristocetin cofactor activity.

This report describes the replacement of the current MFG-scale block operation stages (FIG. 43A) with an improved CAT stage (UNO_S) (FIG. 43B). Improvement in CAT stage has been studied on a small scale. The UDF (concentration/dialysis) step following the CAT step may also need to be optimized.

3. Materials and methods.

This document describes the materials and methods, as well as the sampling plan.

3.1. rVWF download materials.

Frozen MUQ-E was used for all experiments. The material was stored frozen at <-60°C in 130 ml aliquots and thawed overnight at +2 to +8°C as needed. After thawing the MUQ eluate, S/D reagents were added and the mixture was filtered through a 0.2 µm KA02EAVP2S® filter from Pall. The filtered material was then incubated at moderate control for 60 min at room temperature (about +25°C) to inactivate/dissolve potential lipid-enveloped viruses. The S/D reaction was stopped by a 1:2 dilution with 60 mM Na-citrate buffer, pH 7.5. The diluted material was used as a stock solution for the next CAT step.

3.2. Chromatographic equipment.

An AKTA pure 25 small chromatography system (GE Healthcare) was used for the experiments described in this report. The system was equipped with sensors for online monitoring of UV absorption, conductivity, pressure, temperature and pH with electronic recording. The system was controlled by software running Unicorn 7.0. All experiments were carried out at room temperature.

The AKTA system tubing was made from polyetheretherketone (PEEK), which differs from large-scale production that uses the Millipore process system with stainless steel tubing. All hardware components are qualified research and development equipment.

The laboratory column used for all five experiments was equipped with 10 μm polypropylene inlet filters; The UNO_Sphere S resin particle size was approximately 80 µm in diameter. In large scale production, stainless steel input filters with a mesh size of 20 microns are used. All columns are qualified items intended for R&D purposes.

A hardware comparison between the current GEN 1 MGF hardware in the NE and the small-scale GEN 2 hardware used in the current study is shown in FIG. 44.

3.3. Buffers.

Buffers used for small purification runs were manufactured in the laboratory or obtained from the production area. Qualified chemicals were used to prepare the buffers and were also used to produce buffers for experimental clinical production. Buffers were filtered to 0.2 μm pore size and stored in bags or glass vials at room temperature before use. A description of the buffer composition is shown in Fig. 48.

3.4. Analytical methods.

rVWF biochemical characteristics, activity and impurity assays performed include those specific to VWF assay: RistoCo activity, VWF antigen, VWF-propeptide antigen content, FVIII activity chromogenic method, UV absorbance profile (280 nm, 254 nm), polypeptide structure, such as degradation, multimer structure, and CHO NSR content. In some cases, other analytical tests may be performed to determine, but are not limited to, pro-VWF antigen content, FVIII antigen content, furin activity, furin antigen, total protein (BCA), free sulfhydryl, CHO BIP WB, CHO DNA, mouse monoclonal antibody , soy peptone, Triton X-100, polysorbate 80, tri-n-butyl phosphate, dynamic light scattering (DLS) (hydrodynamic radius), sialic acids, n-glycan content, VWF collagen binding and VWF oxidation.

4. Changes to the CAT rVWF 2nd generation (GEN 2) process.

To replace the SEC step in the downstream process of 2nd generation rVWF, the parameters listed below were investigated. Most of the changes made are based on R&D feasibility studies. The type of chromatography resin (UNO_Sphere S resin from BioRad) and the composition (not pH) of the applied buffer did not change. The 2nd generation CAT process included the following changes: S/D processing, concentration and flow rate loading, and wash and elution steps.

4.1. S/D processing.

The S/D treatment was carried out in the same manner as in the GEN 1 process, except that the S/D inactivation was stopped by diluting the virus-inactivated material 1:2 with 60 mM Na citrate buffer, which adjusted the CAT feed to the preferred pH of 7.5 -8.0 (pH test range 6.0-9.0) and to a preferred conductivity of 10-30 mS/cm at +25°C (conductivity test range 5-40 mS/cm ² at +25°C). In the rVWF GEN 1 CAT stage that was performed, the CAT load was set to pH 8.9-9.2. In the GEN 2 setup, conductivity and pH were set at a level that minimized binding of the propeptides CHO-HCP, CHO-DNA, and rVWF to the matrix. Similarly, low molecular weight (LMW) rVWF molecules were prevented from binding to the column matrix, whereas only high molecular weight (HMW) rVWF molecules were preferentially captured. One of the goals of this study was to increase conductivity during the loading phase and remove as much of the rVWF LMW, CHO-HCP, CHO-DNA, and rVWF propeptide from the feed solution as possible.

4.2. Load concentrations and flow rates.

The loading concentration (ME rVWF/mL resin) was increased during the study to provide higher product loading without increasing column volume. Manufacturing scale (MFG) typically uses a resin concentration of 60-140 IU/mL, in contrast, the current small scale study loaded 90-270 IU/mL of resin. The equilibration, loading, and re-equilibration flow rates of the 2nd generation CAT procedure were the same as those of the 1st generation process (100 cm/h). The washing and elution rates were varied as shown in FIG. 45.

4.3. Load concentrations and flow rates.

The washing step preceding the elution phase has been modified to optimize the removal of process and product related impurities. The step elution used in the 1st generation process has been changed to gradient elution. The study examined the length

- 72 045553 gradient. The change in elution procedure was based on the observation that low molecular weight rVWF molecules elute in the early gradient fractions, whereas high molecular weight rVWF molecules elute in the late gradient fractions (see e.g. US

6465624).

5. Comparison of CAT Gen 1 and Gen 2 processes.

In both procedures, the CAT process includes the following steps: column activation (loading the anionic ligand with the cationic sodium counterion) and equilibration (preparing the column for loading in terms of stable pH and conductivity controlled at the column outlet), followed by loading the S/D-treated product and diluted MUQ eluate.

During loading of the MFG scale, the eluate was filtered online through a 0.2 μm filter to protect the column from particulates that may have formed during S/D processing. In the small-scale process this step was skipped. After the solution containing the product was pumped into the column, loading was completed and loosely bound impurities were removed by applying a wash step that removed the low molecular weight S/D reagents that had been pumped into the column. The pH and conductivity of the washing stage correspond to the parameters of the equilibration and loading stage. After washing, bound proteins were eluted from the column by stepwise elution using an elution buffer with increased conductivity and counterion concentration. The product pool was collected with <3.6 TC.

In the small scale Gen 2 process, an alternative gradient elution procedure was used to remove rVWF propeptide, low molecular weight rVWF molecules, and high molecular weight molecules from the column (FIG. 45). The pre-elution wash steps were performed as a step wash with the same pH and conductivity as the starting point of the gradient elution. Four wash scenarios were tested: 0% B (10 OK), 55% B (10 OK), 40% B (5 OK), and 45% B (5 OK) and 36% B (5 OK). The corresponding gradient elution steps were 0-100% B (12 OK), 55-100% B (6 OK), 45-100% B (6 OK), and 36-100% B (6 OK). Elution was completed by washing with 2-3 OK 100% B.

Eluates were pooled based on impurities associated with the product being eluted and product subspecies. After product elution, the column was cleaned and disinfected with alkaline and acidic solutions. The primary purpose of this polishing step was to further remove process related impurities including host cell protein CHO, human rfurin, media compounds such as soy peptone), product related impurities (rVWF propeptide) and low molecular weight S/D reagents. mass. Only a minor effect on rFVIII clearance was expected. After the improved CAT step (UNO_S), a protein concentration and buffer exchange step (ultra/diafiltration) may be required. However, this ultra/diafiltration step was not part of the study described here. The differences in chromatographic procedure between the large scale 1st generation MFG process and the small scale 2nd generation process are shown in FIG. 46-48.

6. Results.

The following section presents the results of the current study. Five experiments carried out on a small scale clearly show that replacing the SEC unit operation step and the upstream ultrafiltration (buffer exchange) step is possible by introducing a modified UNO procedure (CAT).

6.1. Chromatograms.

As stated above, five experiments were performed with different washing and gradient elution procedures. The goal of the UNO S step was to find the optimal method to remove product and process related impurities on the one hand and to achieve the optimal yield in terms of Ag VWF and activity. In fig. Figure 49 shows two chromatograms of the last (5th) cycle of VW_USS_05. On the top panel of Fig. 49 shows the complete cycle including column activation, loading phase (high UV absorbance at 280 nm caused by S/D chemicals in the feed), re-equilibration, wash, gradient elution, 2M NaCl wash and CIP procedure. The chromatogram is combined from 2 result files that explain the x-axis scale (result file 1: activation to end of load; result file 2: start re-equilibration, 36% B wash, gradient elution, CIP). In the bottom panel of Fig. 49 details the elution phase (a wash step to 36% elution buffer B, followed by a gradient elution from 36% B to 100% B and a 100% elution buffer B phase).

6.2. SDS-PAGE.

By changing and optimizing the applied chromatographic conditions (e.g., wash buffer conductivity, start of gradient elution), the separation of propeptide and mature rVWF was refined. Additionally, removal of process-related impurities as well as mature rVWF Ag yield and activity were improved. The results of SDS-PAGE (silver staining and Western blotting for rVWF) of the last (5th) run in the series of experiments are presented in Fig. 50.

SDS-PAGE was performed on 3–8% Tris-acetate gels under reducing conditions. The separated polypeptides were visualized by silver staining (top) and Western blotting (bottom). Before

- 73 045553 loading samples were reduced with DTT, after which free sulfhydryl groups were blocked with iodoacetamide. For Western blotting, the 1st antibody was a polyclonal rabbit anti-human VWF antibody (from Dako; order number A0082; diluted 1:1000), the 2nd antibody was a polyclonal, AP-conjugated goat anti-rabbit IgG antibody (from Sigma; order number A-8025; dilution 1:2000). The rVWF range is greater than 250 kDa; The VWF propeptide has a mass of about 90 kDa. The propeptide is not detected by antibodies used for Western blotting.

The results from the VWF_USS_05 run show a clear separation of the propeptide and mature rVWF. The eluate sample (lane 16) and the reference sample purified according to the generation 1 procedure (lane 18) are very comparable.

6.3. Multimeric analysis.

Agarose gel and Western blot analysis were performed to evaluate the distribution of high and low molecular weight rVWF subspecies multimers. Samples from load, flow through (FT), wash, elution and high salt wash were tested (FIG. 51). LMW rVWF subspecies are contained in the flow-through (FT; effluent fraction) (lane 8) and wash/pre-elution (lanes 9 and 10). The elution and post-elution pools (lanes 11 and 12) show a banding pattern comparable to the SEC-F reference sample (lane 15). The control sample was purified according to the generation 1 (Gen 1) process and approximately corresponds to the increasing peak of the SEC eluate pool. The high-salt wash (lane 13) contains ultra-large rVWF molecules, as can be seen in the smear at the top of the lane.

Multimeric assays were performed on 1% agarose gels using a standard protocol. Approximately 50 ng of rVWF was applied per lane and separated under non-reducing conditions in the presence of urea. The separated polypeptides were visualized by Western blotting using rabbit anti-human VWF antibody (Dako) as the 1st antibody (1:1000 dilution) and AP-conjugated goat anti-rabbit IgG antibody (Sigma) as the 2nd antibody (1:1000 dilution). 2000).

By comparing the distribution of rVWF multimers between the UNO_S (Gen 2) and SEC (Gen 1) runs, the effect of reverse partitioning can be clearly seen. In the SEC procedure, the ultra-large and large molecules (empty volume) are eluted first, followed by the target molecules and the propeptide. In Gen 2, the order of division is exactly the opposite (from small to large). However, both methods resulted in the same distribution of rVWF multimer in the eluate pool. After the UNO_S step, a UDF (concentration/dialysis) operation was required to concentrate the target molecule and transfer it to the formulation buffer.

6.4. Analytical results.

A summary of the analytical results is presented in Fig. 52-55. Each table shows the results of one specific analysis and contains data from all 5 runs performed in the study. A comparative overview of the eluate results is also presented in FIG. 56. In addition to the rVWF:Ag percentage and Risto Co activity of the eluate yield, the table contains calculated rates allowing direct comparison of different run settings.

6.5. Aligning analytics with success criteria.

The target parameters of the eluate (product fraction) obtained from the modified CAT (UNO_S) stage of operation partially correspond to the selected BDS product specifications. Since the CAT-E product pool must be concentrated and dialized to produce BDS material, the design targets (FIG. 57) are essentially ratios (calculated) that are independent of the absolute parameter concentrations.

The fact that most of the development goals were achieved or nearly achieved demonstrates the feasibility of the proposed procedure described in this paper. Not all analytical analyzes were performed, but key results such as rVWF:Ag and Risto yield, removal of CHO NCP and propeptide impurities, and rVWF multimer distribution demonstrate comparable performance of the proposed new CAT procedure and the previously used UNO_S/SEC combination.

7. Discussion.

In this study, five runs of UNO_S were performed to examine the 2nd generation CAT procedure. Results from the optimized (latest) run show separation of high and low molecular weight rVWF multimers as well as removal of rVWF propeptides and CHO-HCP impurities from the target protein, which is comparable to results achieved with the Gen 1 procedure (e.g. UNO_S in stepwise elution mode+ SEC stage). An introduced wash step with a conductivity of about 24 mS/cm (36% elution buffer B) followed by a gradient elution step to about 50 mS/cm (100% elution buffer B) resulted in a CAT eluate pool comparable in quality to the previously obtained Gen 1 pool SEC F. Although an additional UDF step can be used to concentrate and dialyze the CAT eluate, the CAT Gen 2 procedure described herein shows great potential to replace the UDF and SEC work steps used in the downstream Gen 1 process to produce BDS material.

Example 13. Western blot assessment of DF3338/042 and DF3362/023 multimers against VWF.

Mat-rVWF obtained by this method was analyzed for multimeric content. The advantages of cation descent methods (CEX) include:

- 74 045553 reduction in the number of operations - 1 CEX replaces 3 operations of the current process;

removal of the r-vWF propeptide and removal of host cell proteins are similar to the affinity step.

When SD processing is enabled on the cation exchanger column 4, the operations are included in one step.

Including SD treatments on the column and furin maturation on the cation exchanger, 5 operations are included in one step.

Reduced shear stress, which reduces the risk of thrombotic rVWF formation (due to fewer operations, filtration and significantly reduced retention time).

Western blotting was performed for this analysis. Western blot images were imported into Corel Photo Paint Software and converted to 16-bit gray scale images. 16-bit gray scale format is a requirement for evaluation. Evaluation was carried out using Image Quant 1D software.

To simplify evaluation, the images were flipped vertically (track numbers remained the same):

lanes 1 -6=low molecular weight;

bands 7-12=average molecular weight;

bands >12=high molecular weight.

Densitometric assessment of vWF multimers of a product derived from an improved CEX as described herein, compared to a product obtained from a 3-step process.

Table 11

Densitometric evaluation results

Benchmark vw_uss_o 4E VW_USS_0 5E % with low MM SUM bands 1-6 40.86 34.91 38.39 % with average MM SUM bands 7-12 40.27 39 36.87 % with high MM SUM bands > 12 18.87 26.08 24.74

Raw data showing the percentage of multimer is presented in FIG. 61-63.

Example 14: Variant of the vWF cleaning process.

I. State of the art.

The r-vWF propeptide is a product-bound contaminant of the r-VWF product derived from CHO cells. The production cell line generates r-VWF, which contains about 60% pro-r-vWF. The r-VWF propeptide is attached to the r-vWF polypeptide covalently by a peptide amide bond and additionally non-covalently by divalent cations. The covalent peptide amide bond is cleaved by in vitro incubation with rfurin. However, the cleaved r-VWF propeptide remains attached to the VWF molecule, and this example describes a method for separating the two polypeptides. The rvWF/rvWF_PP complex was found to be stabilized by divalent cations and low pH. By using a divalent cation chelator or high pH in combination with a proper separation method, the two molecules can be separated with high efficiency and reliability. Because a chelating agent, low concentrations of EDTA or citrate are found to be effective and a pH greater than or equal to pH 7 is also considered effective when applied to a cation exchange resin as a wash procedure or in size exclusion chromatography when applied to a separation buffer. The same principle should apply to all separation technologies, including ion exchange or size separation using resins or membrane technology. In the current manufacturing process for rVWF, the SEC step is performed with a running buffer containing citrate to support the separation of rVWF and rVWF-PP.

1. Application example description - VW_USS_07.

1. Removal of r-vWF propeptide.

2. Example of alternative processing of SD_VI on a column.

3. Creation of the rFVIII/r-vWF complex on a column.

4. Pre-formulation on the column while eluting the rFVIII/r-vWF complex in an alternative composition buffer system.

Process details.

After the monoclonal antibody step to capture recombinant factor VIII, the flow buffer containing r-vWF is loaded onto a Fractogel TMAE anion exchanger. r-vWF is on the anion exchanger and matures with furin in the presence of calcium. r-vWF was eluted from the anion exchanger with increasing conductivity. The TMAE eluate was filtered through a Mustang Q filter unit (Mustang Q, Pall part number XT5000MSTGQP1) to remove CHO-DNA and impurities that bind to

- 75 045553 filter membrane. The product containing MUQ_flow buffer was treated by dilution 1:2 [60 mM sodium citrate, pH 7.6] to a conductivity of 21.9 mS/cm and pH 7.16. High conductivity is selected to ensure removal of the r-vWF propeptide and low molecular weight r-vWF to utilize the resin's capacity for the desired high molecular weight r-vWF. The treated loading solution was loaded onto UNOsphere TM S cation exchange medium (Bio Rad, cat. no.: 156-0115) with internal diameter = 10 mm, layer height 8.8 cm, volume 6.91 ml with a flow rate of 100 cm/h, followed by first wash. (Re-equilibrate) 2CV with [30 mM Na-citrate, 180 mM NaCl, pH 7.5] to remove tightly bound HCP and r-vWF propeptide.

Potential on-column treatment (WSD) was performed with [30 mM Na-citrate, 180 mM NaCl, pH 7.5, containing 25 g/kg mixture of 18.0 g polysorbate 80, 3.5 g dimethyl sulfoxide DMSO, 3.5 g TnBP ] in column 12. Volumes and contact times were approximately 1 hour to inactivate viruses in the lipid envelope. The on-column treatment components were washed with wash 2 in 10 column volumes [30 mM Na-citrate, 180 mM NaCl, pH 7.5]. Using wash 3, the buffer was changed from a sodium citrate buffer system to a glycine/taurine system using [50 mM glycine, 10 mM taurine, 10% sucrose, 0.1% polysorbate 80, pH 5.5] in 4 column volumes. In the FVIII-Con step, recombinant human factor VIII obtained from the ADVATE process was loaded onto bound r-vWF in 10 column volumes.

FVIII-Con buffer consists of [1.57 g rFVIII S2 ADV S17B010901B2 diluted in 218.67 g 50 mM glycine, 10 mM taurine, 5% (w/w) sucrose, 5% (w/w) D-mannitol, 0.1% polysorbate. 80.2 mM CaCl ₂ , 150 mM NaCl and pH 7.4]. Wash 4 was used to wash away unbound rFVIII and to prepare the pre-specimen buffer matrix by loading 5 column volumes [50 mM glycine, 10 mM taurine, 5% (w/w) sucrose, 5% (w/w) D -mannitol, 0.1% polysorbate 80, 2 mM CaCl ₂ , 150 mM NaCl, pH 7.4]. Both r-vWF and rFVIII were eluted [50 mM glycine, 10 mM taurine, 5% (w/w) sucrose, 5% (w/w) D-mannitol, 0.1% polysorbate 80, 2 mM CaCl ₂ , 600 mM NaCl, pH 7.4 ± 0.2] from the column to form an eluate. The eluate was diluted to adjust the sodium chloride content to approximately 150 mM NaCl [50 mM glycine, 10 mM taurine, 5% (w/w) sucrose, 5% (w/w) D-mannitol, 0.1% polysorbate 80.2 mM CaCl ₂ , pH 7.4].

Sequence of the process.

The sequence of key steps in this example consists of the following steps (see also the bottom of Fig. 67 for the chromatography diagram).

1. Capture Mab FVIII (FT is the fraction containing r-vWF).

2. Fractogel TMAE capture + maturation.

3. Mustang Q in FT mode.

4. SEX, as described (VW_USS_07).

Result.

The experiment was successfully carried out at all 4 points.

1. Removal of the r-vWF propeptide occurred during Wash 1, WSD (Wash with (S)olvent (D)detergent) and Wash 2 steps (see FIG. 67 , 68 and 69).

2. Example of alternative on-column processing of SD_VI at the WSD stage.

3. Creation of the rFVIII/r-vWF complex on a column - FVIII-Con stage.

4. Pre-formulation on the column while the rFVIII/r-vWF complex is eluted in an alternative composition buffer system (see FIG. 66, last row).

Example 15: A variation of the vWF purification process - sialylation testing.

I. State of the art.

The r-vWF propeptide is a product-bound contaminant of the r-VWF product derived from CHO cells. The production cell line generates r-VWF, which contains about 60% pro-r-vWF. The r-vWF propeptide is attached to the r-VWF polypeptide covalently by a peptide amide bond and additionally non-covalently by divalent cations. The covalent peptide amide bond is cleaved by in vitro incubation with rfurin. However, the cleaved r-VWF propeptide remains attached to the VWF molecule, and this example describes a method for separating the two polypeptides. This example provides an alternative embodiment for separating the r-vWF propeptide from the rVWF polypeptide after furin cleavage to test for additional sialylation. Additional details and results of the purification process are depicted in FIG. 70-73 and 78.

1. Experiment No.: VW_USS_06.

1. Removal of r-vWF propeptide.

2. Carrying out additional 2,6-sialylation on a column with r-vWF.

2. Experiment No.: VW_USS_06.

After the monoclonal antibody step to capture recombinant factor VIII, the flow buffer containing r-vWF is loaded onto a Fractogel TMAE anion exchanger. r-vWF is on the anion exchanger and matures with furin in the presence of calcium. r-vWF was eluted from the anion exchanger with increasing conductivity. The TMAE eluate was filtered through a Mustang Q filter unit (Mustang Q, Pall part number XT5000MSTGQP1) to remove CHO-DNA and impurities that bound to the filter membrane. The product containing MUQ_flow buffer was processed by dilution 1:2

- 76 045553

[60 mM sodium citrate, pH 7.6] to a conductivity of 18.39 mS/cm and pH 7.33. High conductivity is selected to ensure removal of the r-vWF propeptide and low molecular weight r-vWF to utilize the resin's capacity for the desired high molecular weight r-vWF. The treated loading solution was loaded onto UNOsphereTM S cation exchange medium (Bio Rad, cat. no.: 156-0115) with internal diameter = 10 mm, layer height 8.8 cm, volume 6.91 ml with a flow rate of 100 cm/h, followed by the first washing. (Re-equilibrate) 2CV with [30 mM Na-citrate, 180 mM NaCl, pH 7.5] to remove tightly bound HCP and r-vWF propeptide. To introduce additional 2,6 sialylation, a mixture of 50% (v/v) CMP-NANA solution based on [30 mM Na-citrate, 180 mM NaCl, pH 7.5] and 50% (v/v) alpha 2 ,6-sialyltransferase based on [30 mM Na-citrate, 180 mM NaCl, pH 7.5] was applied to the column in 10 column volumes and at a flow rate of 25 cm/h by continuous stirring. CMP-NANA solution composition: 11 mg CMP_NANA C8271-25 mg lot number: SLBV 7777 dissolved in 154.29 g [30 mM Na-citrate, 180 mM NaCl, pH 7.5]. The alpha-2,6-sialyltransferase buffer contained alpha-2,6-sialyltransferase S2076-1UN SIGMA, lot number SLBV0552 from Photobacterium damsela, dissolved in 1 ml of purified water - 0.5 g of dissolved alpha-2,6-sialyltransferase was diluted 152.10 g [30 mM Na-citrate, 180 mM NaCl, pH 7.5]. An additional wash with 2 column volumes [30 mM Na-citrate, 180 mM NaCl, pH 7.5] was used to remove excess CMP_NANA and alpha-2,6-sialyltransferase. Buffer exchange was achieved by applying 4 column volumes [50 mM HEPES, 150 mM NaCl pH 6.0]. Elution was performed with [50 mM HEPES, 500 mM NaCl, pH 7.5] in 4 column volumes.

3. Complete cleaning sequence VW_USS_06.

The sequence of key stages in this example consists of the following stages.

1. Capture Mab FVIII (FT is the fraction containing r-vWF).

2. Fractogel TMAE capture + maturation.

3. Mustang Q in FT mode.

4. SEX as described (VW_USS_06).

Result.

No additional 2,6-sialylation was detected using the method in this example. However, 2,3-sialylation was detected, which is a common sialylation pattern for rvWF.

Example 16: A variation of the vWF purification process - sialylation testing.

I. State of the art.

The r-vWF propeptide is a product-bound contaminant of the r-VWF product derived from CHO cells. The production cell line generates r-VWF, which contains about 60% pro-r-vWF. The r-vWF propeptide is attached to the r-VWF polypeptide covalently by a peptide amide bond and additionally non-covalently by divalent cations. The covalent peptide amide bond is cleaved by in vitro incubation with rfurin. However, the cleaved r-VWF propeptide remains attached to the VWF molecule, and this example describes a method for separating the two polypeptides. This example provides an alternative embodiment for separating the r-vWF propeptide from the rVWF polypeptide after furin cleavage to test for additional sialylation. Additional details and results of the purification process are depicted in FIG. 74-78.

1. Experiment No.: VW_USS_08.

1. Removal of r-vWF propeptide.

2. Carrying out additional 2,6-sialylation on a column with r-vWF.

2. Experiment No.: VW_USS_08 E.

After the monoclonal antibody step to capture recombinant factor VIII, the flow buffer containing r-vWF is loaded onto a Fractogel TMAE anion exchanger. r-vWF is on the anion exchanger and matures with furin in the presence of calcium. r-vWF was eluted from the anion exchanger with increasing conductivity. The TMAE eluate was filtered through a Mustang Q filter unit (Mustang Q, Pall part number XT5000MSTGQP1) to remove CHO-DNA and impurities that bound to the filter membrane. The product containing MUQ_flow buffer was treated by diluting 1:2 [60 mM sodium citrate, pH 7.6] to a conductivity of 19.97 mS/cm and pH 7.33. High conductivity is selected to ensure removal of the r-vWF propeptide and low molecular weight r-vWF to utilize the resin's capacity for the desired high molecular weight r-vWF. The treated loading solution was loaded onto UNOsphereTM S cation exchange medium (Bio Rad, cat. no.: 156-0115) with internal diameter = 10 mm, layer height 8.8 cm, volume 6.91 ml with a flow rate of 100 cm/h, followed by the first washing. (Re-equilibrate) 2CV with [30 mM Na-citrate, 180 mM NaCl, pH 7.5] to remove tightly bound HCP and r-vWF propeptide. To introduce additional 2,6 sialylation, a mixture of 50% (v/v) CMP-NANA solution based on [30 mM Na-citrate, 180 mM NaCl, pH 7.5] and 50% (v/v) alpha 2 ,6-sialyltransferase based on [30 mM Na-citrate, 180 mM NaCl, pH 7.5] was applied to the column in 10 column volumes and at a flow rate of 25 cm/h by continuous stirring. The composition of the CMP-NANA solution was 14 mg CMP_NANA C8271-25 mg Lot No. SLBV 7777 dissolved in 121.57 g [30 mM Na-citrate, 180 mM NaCl, pH 7.5]. The composition of the buffer for al

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Claims (24)

alpha-2,6-sialyltransferase included alpha-2,6-sialyltransferase S2076-1UN SIGMA, lot number SLBV0552 from Photobacterium damsela was dissolved in 121.10 g [30 mM Na-citrate, 180 mM NaCl, pH 7.5]. An additional wash with 2 column volumes [30 mM Na-citrate, 180 mM NaCl, pH 7.5] was used to remove excess CMP_NANA and alpha-2,6-sialyltransferase. Buffer exchange was achieved by applying 4 column volumes [50 mM HEPES, 150 mM NaCl pH 6.0]. Elution was performed with [50 mM HEPES, 500 mM NaCl, pH 7.5] in 4 column volumes. 3. Complete cleaning sequence VW_USS_08. The sequence of key stages in this example consists of the following stages. 1. Capture Mab FVIII (FT is the fraction containing r-vWF). 2. Fractogel TMAE capture + maturation. 3. Mustang Q in FT mode. 4. SEX as described (VW_USS_08). Result. No additional 2,6-sialylation was detected using the method in this example. However, 2,3-sialylation was detected, which is a common sialylation pattern for r-vWF. The above examples are provided to provide those skilled in the art with complete disclosure and description of how to make and use embodiments of the compositions, systems and methods of the invention, and are not intended to limit the scope of what the inventors believe to be their invention. Modifications to the above-described methods of carrying out the invention, which are obvious to those skilled in the art, are included in the scope of the following claims. All patents and publications mentioned in the description indicate the level of qualification of specialists in the field of technology to which the invention relates. All references cited herein are incorporated by reference to the same extent as if each reference had been incorporated by reference in its entirety. All headings and section designations are used for clarity and reference purposes only and should not be construed as limiting in any way. For example, those skilled in the art will appreciate the usefulness of combining various aspects from different headings and sections in accordance with the spirit and scope of the invention described herein. All references cited herein are hereby incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication, patent, or patent application had been specifically and individually indicated to be incorporated by reference. in its entirety for all purposes. Many modifications and variations of the present invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments and examples described herein are offered by way of example only, and application is to be limited only by the terms of the appended claims together with the full scope of equivalents covered therein. CLAIM 1. A method for producing a composition containing highly purified mature recombinant von Willebrand factor (rVWF) devoid of propeptide (mat-rVWF), wherein said method includes the steps of: providing a solution containing the mat-rVWF/rVWF-PP complex, mat-rVWF and rVWF propeptide (rVWF-PP); inducing dissociation of the specified mat-rVWF/rVWF-PP complex in the specified solution into a) onto mat-rVWF and rVWF-PP, wherein said dissociation occurs by destruction of non-covalently bound mat-rVWF and rVWF-PP, wherein said dissociation is induced by: adding at least one chelating agent, or increasing the pH to a pH of at least 7; and collecting said mat-rVWF to obtain a highly purified mat-rVWF composition, wherein said highly purified mat-rVWF composition contains at least 95% mature rVWF and less than 5% rVWF-PP, wherein said collection in step b) of claim 1 includes one or more protein separation methods selected from the group consisting of ion exchange chromatography (IEC), size exclusion chromatography (SEC), size separation by membrane technology, and affinity chromatography. 2. The method according to claim 1, in which the composition of highly purified mat-rVWF contains at least 96% mat-rVWF and less than 4% rVWF-PP, at least 97% mat-rVWF and less than 3% rVWF-PP, at least 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% rVWFPP. 3. The method of any one of the preceding claims, wherein said solution is selected from the group consisting of a cell culture medium, an antibody column flow solution, and - 78 045553 buffered solution. 4. The method according to any of the preceding claims, wherein said solution is treated with furin before step a). 5. The method according to any of the preceding claims, wherein said at least one chelating agent is a divalent cation chelating agent. 6. The method of claim 5, wherein said divalent cation chelating agent is selected from the group consisting of EDTA, EGTA, CDTA and citrate. 7. The method according to any of the preceding paragraphs, in which the specified 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. 8. The method of any one of the preceding claims, wherein said pH is increased from at least about 7.2 to about 7.8. 9. The method of any one of the preceding claims, wherein said pH is increased to at least about 7.6. 10. A method according to any one of the preceding claims, wherein said pH is increased by adding basic amino acids, Tris, NaOH, Tricine or ethanolamine. 11. The method of claim 1, wherein said protein separation method is size exclusion chromatography (SEC). 12. The method of claim 1, wherein said one or more protein separation methods are ion exchange chromatography (IEC). 13. The method of any one of the preceding claims, wherein said one or more protein separation methods comprise a buffer system, wherein said buffer system comprises one or more buffers. 14. The method according to claim 13, in which the specified buffer system is selected from the group consisting of glycine HEPES (4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid), tris-HCl (tris(hydroxymethyl)-aminomethane), histidine, imidazole, citrate acetate, and MES (2-(N-morpholino)ethanesulfonic acid). 15. The method of claim 13 or 14, wherein said buffer further contains one or more monovalent cations. 16. The method of claim 15, wherein said one or more monovalent cations are selected from the group consisting of Na ⁺ , K ⁺ , Li ⁺ and Cs ⁺ . 17. The method of claim 15, wherein said monovalent cation is Na ⁺ . 18. The method according to any one of claims 13 to 17, wherein said buffer further contains one or more monovalent, divalent and/or trivalent anions. 19. The method according to claim 18, wherein said one or more monovalent, divalent and/or trivalent anions are selected from the group consisting of Cl ^- , acetate ^- , SO ₄ ² - , Br ^- and citrate ^{3 -} . 20. The method according to any one of claims 13 to 19, wherein said buffer system contains at least one buffer having a conductivity of >0.5 mS/cm at 25°C. 21. The method according to any one of claims 13 to 20, wherein said buffer system contains at least one buffer having a conductivity of 15.0 ± 0.2 mS/cm at 25°C. 22. The method of any one of the preceding claims, wherein said highly purified mat-rVWF composition contains a host cell impurity (HC) level of <2.0%. 23. The method of any one of the preceding claims, wherein said highly purified mat-rVWF composition contains a host cell impurity (HC) level of <0.6%. 24. The method according to any one of the preceding claims, wherein said highly purified mat-rVWF composition is lyophilized after purification. -