WO2013098676A1

WO2013098676A1 - Method for purifying factor viii

Info

Publication number: WO2013098676A1
Application number: PCT/IB2012/056802
Authority: WO
Inventors: Dean CHAMBERLAIN
Original assignee: Grifols SA
Current assignee: Grifols SA
Priority date: 2011-12-30
Filing date: 2012-11-28
Publication date: 2013-07-04
Anticipated expiration: 2014-06-30
Also published as: AR089245A1; UY34501A; AU2012324020A1

Abstract

The invention relates to a method for the purification of FVIII by affinity chromatography characterized in that a composition comprising FVIII is contacted with an affinity ligand, in particular an anti-FVIII antibody, under such conditions that FVIII binds to the affinity ligand, but not to any vWF that may or may not be present in the composition.

Description

METHOD FOR PURIFYING FACTOR VIII FIELD OF THE INVENTION

The invention relates to a method for purifying Factor VIII (FVIII) from a starting material by affinity chromatography, as well as compositions comprising the purified FVIII.

BACKGROUND OF THE INVENTION

Haemophilia A is characterized by the lack or insufficient function of FVIII. Patients with severe haemophilia A, may be administered recombinant or plasma-derived FVIII as a replacement therapy. Human FVIII, a 330 kD glycoprotein, circulates in plasma, mainly in the form of a non-covalently bound complex with von Willebrand factor (vWF) .

Processes for the purification of FVIII are known, including using immobilized antibodies to human von Willebrand factor (vWF) in which vWF/FVIII complexes are adsorbed. The FVIII that is subsequently purified away from the FVIII/vWF complex may be contaminated by vWF . Thus, one problem in the recovery of such a complex particularly is the separation of molecules containing vWF, and the enrichment for the FVIII.

There remains a need for a method for the purification of FVIII.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a 7% Tris-Acetate gel showing a purification process of plasma-derived FVIII using VlllSelect (GE Healthcare, Piscataway, NJ) (Catalog No. 17-5450), as described in Example 2. Lane 1: Load = 15.6 U/mL (50mL total); Lane 2: Elution (no additional CaCl₂) = 4.7U/mL (4mL total); and Lane 3: Elution (400mM additional CaCl₂) = 43.lU/mL (4mL total).

Figure 2 is a 7% Tris-Acetate gel showing a purification process of recombinant FVIII using VlllSelect (GE Healthcare, Piscataway, NJ) (Catalog No. 17-5450), as described in Example 3. Lane 1: ~10 I.U. Xyntha® Antihemophilic Factor ( recombinant ly produced in non-human cell lines) (Wyeth Pharmaceuticals Inc., Philadelphia, PA) ; Lane 2: Supernatant; Lane 3: Resuspended PEG pellet; Lane 4 Filtered Load; and Lane 5: Flow through. SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for preparing a FVIII. The method comprises contacting the FVIII with an affinity ligand in the presence of an amount of a doubly charged ion, wherein the amount of the doubly charged ion is sufficient for the FVIII to form a complex with the affinity ligand but not a vWF .

In another aspect, the present invention provides a composition comprising a FVIII bound to an affinity ligand in the presence of an amount of a doubly charged ion, wherein the amount is sufficient to prevent binding of a vWF to the FVIII.

In other aspects, the present invention provides an affinity ligand equilibrated with an amount of a doubly charged ion; and having a FVIII bound thereto, wherein the amount of the doubly charged ion is sufficient for the FVIII to form a complex with the affinity ligand but not a vWF . Compositions comprising FVIII as purified by the methods of the invention also are provided in still further aspects.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides a method for preparing a FVIII by affinity chromatography. The method comprises contacting the FVIII with an affinity ligand in the presence of an amount of a doubly charged ion, wherein the amount of the doubly charged ion is sufficient for the FVIII to form a complex with the affinity ligand but not a vWF .

In some embodiments, the doubly charged ion is a divalent cation such as, for example, Ca⁺⁺ . In one embodiment, the affinity ligand is an anti-FVIII antibody .

In another embodiment, the doubly charged ion is a calcium ion .

In some embodiments, a composition comprising the FVIII is contacted with an anti-FVIII antibody in the presence of an amount of calcium ions sufficient to allow the anti-FVIII antibody to specifically bind to the FVIII, wherein, if the composition further comprises a vWF protein, the amount of calcium ions prevents association of the FVIII with the vWF to form a FVIII/vWF complex.

In a method according to the invention, m some embodiments, the affinity chromatography may be preceded and/or followed by one or more further steps including, but not limited to, e.g., precipitation, filtration, and chromatographic purification .

The present invention includes improved processes for purification of FVIII, either plasma-derived or recombinant, wherein an optimal concentration of doubly charged ions is used .

In some embodiment, the affinity chromatography preferably effected at a pH ranging from 6.0 to preferably at pH 7.4.

In other embodiments, a protemaceous composition comprising a F I I I /vWF-complex, such as, e.g., a plasma fraction, a cryoprecipitate or a cell-free culture supernatant derived from transformed cells is used. In some embodiments, the composition may also be an enriched protein fraction of a chromatographic method.

In other embodiments, the step of contacting comprises: contacting the affinity ligand with a composition comprising (i) the FVIII; (ii) the vWF; and (ii) the amount of the doubly charged ion. In one embodiment, the step of contacting comprises:

(a) contacting the affinity ligand with a composition comprising a FVIII/vWF complex; and

(b) providing the amount of the doubly charged ion.

In another embodiment, the step of contacting comprises:

(a) equilibrating the affinity ligand with the amount of the doubly charged ion; and

(b) contacting the affinity ligand of step (a) with a composition comprising a FVIII/vWF complex.

FVIII

FVIII molecules of human, non-human (e.g., primates, dogs, cats, horses, pigs, mice, rats, guinea pigs, rabbits, cows, other vertebrates), and hybrid origin are contemplated by the present invention including natural, synthetic, and recombinant proteins. Also within the scope of the present invention are FVIII molecules corresponding to wild-type proteins, or mutants, variants, and/or truncations thereof. Non-limiting examples of FVIII amino acid and nucleic acid sequences are disclosed by, e.g., GenBank Accession nos . 1012296A AAA52420.1, CAA25619.1, AAA52484.1, 1012298A, EAW72647.1, EAW72646.1, XP_001498954.1 , ACK44290.1,

AC095359.1, NP_001138980.1, ABZ10503.1, NP_032003.2, U.S. Patent No. 6,307,032, and Wood et al., Nature, 312:330-7 (1984), each of which is herein incorporated by reference for its teaching of FVIII sequences. Variants, derivatives, modifications, and complexes of FVIII also are known in the art, and are encompassed in the present invention. For example, as described in, U.S. Pat. No. 5,668,108 which discloses variants of FVIII whereby the aspartic acid at position 1241 is replaced by a glutamic acid with the accompanying nucleic acid changes as well; U.S. Pat. No. 5,149,637 describes FVIII variants comprising the C-terminal fraction, either glycosylated or unglycosylated; and U.S. Pat. No. 5,661,008 describes a FVIII variant comprising amino acids 1-740 linked to amino acids 1649 to 2332 by at least 3 amino acid residues; each of which is herein incorporated by reference for each teaching of FVIII variant sequences .

The terms "B domain deletion, " "B-domain deleted, " and "BDD," as used herein with respect to FVIII protein, refer to a FVIII protein in which some or all removal of the amino acids between residues 711 and 1694 have been deleted, and which still preserves a biologically active FVIII molecule.

In some embodiment, the FVIII is a B-domain deleted FVIII protein (a/k/a BDD-FVIII) . A number of B domain deletions can exist depending on which amino acid residues in the B domain of FVIII is deleted and whereby the biological activity of the FVIII molecule is still preserved. A specific B domain deletion called the SQN exists which is created by fusing Ser 743 to Gin 1638 (Lind et al . , Eur J. Biochem 323:19-27, (1995); and WO 1991/09122, each of which is herein incorporated by reference for its teaching of B- domain deleted FVIII. This deletes amino acid residues 744 to 1637 from the B domain creating a Ser-Glu-Asn (SQN) link between the A2 and A3 FVIII domains. Recombinant FVIII

In one embodiment, the FVIII is recombinant FVIII, e.g. active human FVIII expressed in cultured mammalian cells from recombinant DNA clones. Expression systems for the production of FVIII are known in the art, and include prokaryotic and eukaryotic cells, as exemplified by WO 2011/060242, U.S. Pat. Nos. 5,633,150, 5,804,420, and 5,422,250, each of which is incorporated herein for its teaching of production of FVIII.

The production of recombinant FVIII can be carried out by methods well known in the art, see e.g., U.S. Patent No. 6,855,544; Wood, W. I. et al. (1984) Nature 312, 330-337; and Toole, J. J. et al . (1984) Nature 312, 342-347, which are herein incorporated by reference for their teaching of methods and compositions for the production of recombinant proteins. FVIII cDNA assembled into an appropriate transcription unit can be introduced into a suitable host organism for expression of the FVIII protein. Preferably this organism should be an animal cell-line of vertebrate origin in order to ensure correct post-translational modifications .

Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651 ), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the CV-l/EBNA-1 cell line derived from the African green monkey kidney cell line CVI (ATCC CCL 70) as described by McMahan et al., EMBO J. 10:2821 (1991). Other suitable cell lines include, but are not limited to, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS-1), human hepatocellular carcinoma cells (e.g., Hep G2), human adenovirus transformed 293 cells, mouse L-929 cells, HaK hamster cell lines, murine 3T3 cells derived from Swiss, Balb-c or NIH mice and a number of other cell lines. Another suitable mammalian cell line is the CV-1 cell line. In one embodiment, recombinant FVI I I is prepared using the PER.C6^® technology (Crucell, Holland, The Netherlands) .

Prior to the affinity chromatography step of the present invention, the recombinant FVI I I protein, which according to some embodiments accumulates in the medium of the cultured cells, may be concentrated and purified by a variety of biochemical methods, including methods utilizing differences in size, charge, solubility, hydrophobicity, specific affinity, etc. between the recombinant FVI I I and other substances in the conditioned medium.

In some embodiments, the recombinant FVI I I is co-expressed with a recombinant vWF to provide a composition comprising a recombinant FVI I I /recombinant vWF complex.

In one embodiment, the recombinant FVI I I that is present in the composition is subjected to the affinity chromatography step of the present invention. Plasma-Derived FVIII

In other embodiments, the FVIII is plasma-derived FVIII. The preparation of FVIII from human plasma can be carried out by methods well known in the art, e.g. as described by Andersson et al., Proc. Natl. Acad. Sci . U.S.A. 83, 2979- 2983 (1986) .

For example, plasma or a plasma fraction can be used as the starting material. For example, a cryoprecipitate can be used as a plasma fraction, possibly after prior adsorption treatment to remove prothrombin complex factors, or an analogous plasma fraction can be used, for example a Cohn fraction like Cohn I, or a corresponding fraction in accordance with, e.g., Pool et al . , New England Journal of Medicine 273:1443-1447 (1965) . Optionally, fibrinogen can be removed by precipitation.

To eliminate or minimize the risk of the transmission of a pathogenic agent including viruses transmitted by blood such as HIV and hepatitis viruses, for example HAV, HBV, HCV, HGV and parvo viruses, and also the infectious causative agents of BSE and CJD, a one or more steps can be carried out. The plasma-derived FVIII, or a complex comprising the FVIII (e.g., a FVIII :C/vWF complex) can be subjected to inactivation or depletion of the human pathogens before and/or after the affinity chromatography. Methods using virucidal chemical substances can be performed, preferably before and/or during a chromatographic step and, as a result, the risk of a virucidal agent can be efficiently minimized or eliminated. In some embodiments, at least two measures that bring about inactivation and/or depletion of the viruses by different mechanisms are employed including, but not limited to, chemical, chemical-physical and physical methods .

Examples of methods for inactivation of pathogens are, for example, treatment with organic solvents and/or detergents, treatment with chaotropic agents, heat treatment methods, preferably in lyophilized dry or wet state, and combinations thereof. The methods of removal of human pathogens include, e.g., filtration using ultrafilters , deep bed filters, and nanofilters .

In some embodiments, the plasma-derived FVIII is recovered as a FVIII/vWF complex.

In one embodiment, the plasma-derived FVIII that is present in the FVIII/vWF complex is subjected to the affinity chromatography step of the present invention.

Affinity Chromatography

Affinity ligand

In some embodiments, an affinity ligand is used, which is selected from the group consisting of antibodies, fragments thereof and derivatives thereof, i.e. affinity ligands based on an immunoglobulin scaffold. The "fragments" and "derivatives" are capable of selective interaction with the same antigen or epitope (s) of the FVIII as the antibody they are fragments or derivatives thereof. Anti-FVIII antibodies comprise monoclonal and polyclonal antibodies of any origin, including murine, human and other antibodies, as well as chimeric antibodies comprising sequences from different species, such as partly humanized mouse antibodies.

Polyclonal antibodies can be produced by immunization of animals with the FVIII antigen of choice, whereas monoclonal antibodies of defined specificity can be produced using the hybridoma technology developed by, e.g., Kohler G and Milstein C, Eur. J. Immunol. 6:511-519 (1976).

Antibody fragments and derivatives comprise Fab fragments, consisting of the first constant domain of the heavy chain (CHI), the constant domain of the light chain (CL) , the variable domain of the heavy chain (VH) and the variable domain of the light chain (VL) of an intact immunoglobulin protein; Fv fragments, consisting of the two variable antibody domains VH and VL (Skerra A and Pluckthun A (1988) Science 240:1038-1041); single chain Fv fragments (scFv), consisting of the two VH and VL domains linked together by a flexible peptide linker (Bird R E and Walker B W (1991) Trends Biotechnol. 9:132-137); Bence Jones dimers (Stevens F J et al. (1991) Biochemistry 30:6803-6805); camelid heavy- chain dimers (Hamers-Casterman C et al. (1993) Nature 363:446-448) and single variable domains (Cai X and Garen A (1996) Proc. Natl. Acad. Sci. U.S.A. 93:6280-6285; Masat L et al. (1994) Proc. Natl. Acad. Sci. U.S.A. 91:893-896), and single domain scaffolds like e.g. the New Antigen Receptor (NAR) from the nurse shark (Dooley H et al . (2003) Mol. Immunol. 40:25-33) and minibodies based on a variable heavy domain (Skerra A and Pluckthun A (1988) Science 240:1038- 1041) .

Anti-FVIII antibodies and/or binding fragments thereof are disclosed by e.g., Jacquemin et al., Blood. (1998) 92:496- 506 and U.S. Patent Publication No. 2008/0206254, each of which is incorporated herein for its teaching of anti-FVIII antibody.

For example, in some embodiments, anti-FVIII antibodies for use in the methods of the invention include antibodies that specifically bind FVI I I antigen, wherein the antibody (a) comprises an antigen binding domain of an antibody disclosed by Jacquemin et al., Blood. (1998) 92:496-506 or U.S. Patent Publication No. 2008/0206254; (b) competes for FVI I I binding with one or more of the antibodies disclosed by Jacquemin et al., Blood. (1998) 92:496-506 or U.S. Patent Publication No. 2008/0206254; (c) binds a FVI I I epitope bound by an antibody disclosed by Jacquemin et al., Blood. (1998) 92:496-506 or U.S. Patent Publication No. 2008/0206254s; or (d) comprises a FVI I I-binding fragment of an antibody of (a) -(c) . In another embodiment, the affinity ligand for use in the methods of the invention is an antibody characterized in that the heavy chain of the variable region of the antibody comprises, in its CDRs, the sequences corresponding SEQ ID NO:l (Gly Asp Ser lie Ser Asp Tyr Tyr Trp Ser) , 2 (Tyr Phe Phe Tyr Ser Gly Gly Ser Asn Tyr Asn Pro Ser Leu Lys Ser) , and 3 (Ser Gin Leu Arg Tyr Tyr Leu Asp Val) or sequences having at least 80% or at least 90% or 95% sequence identity; and the light chain variable region comprises, in its CDRs the sequences of SEQ ID NO : 4 (Ser Gin Ser Val Asp Ser Asn Tyr Leu Ala) , 5 (Gly Ala Ser Asn Arg Ala Thr) , and 6 (Gin Gin Tyr Gly Ser Phe) or sequences having at least 80% or at least 90% or 95% sequence identity therewith.

In other embodiments, the affinity ligand for use in the methods of the invention is an antibody characterized in that the heavy chain variable region comprises the sequence of SEQ ID NO: 7 (Gin Val Gin Leu Gin Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser lie Ser Asp Tyr Tyr Trp Ser Trp lie Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp lie Gly Tyr Phe Phe Tyr Ser Gly Gly Ser Asn Tyr Asn Pro Ser Leu Lys Ser Arg Val Thr Met Ser Val Asp Thr Ser Lys Asn Gin Phe Ser Leu Lys Leu Gly Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Ser Gin Leu Arg Tyr Tyr Leu Asp Val Trp Gly Gin Gly Thr Thr Val Thr Val Ser Ser) or a sequence having at least 80% or at least 90% or 95% sequence identity therewith within the CDR regions and/or the light chain variable region comprises the sequence of SEQ ID NO : 8 (Glu lie Val Leu Thr Gin Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Asp Ser Asn Tyr Leu Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ala Pro Arg Val Val lie Tyr Gly Ala Ser Asn Arg Ala Thr Gly lie Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr lie Ser Arg Leu Asp Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Tyr Gly Ser Phe Phe Gly Gin Gly Thr Arg Leu Glu lie Lys) or a sequence having at least 80% or at least 90% or 95% sequence identity therewith within the CDR regions.

In some embodiments, the affinity ligand for use in the methods of the invention is the antibody designated as BOIIB2 produced by the cell line deposited with the Belgian Coordinated Collections of Microorganisms (BCCM) under deposit Accession No. LMBP 6422CB, capable of specifically binding to the A2 domain of FVIII; and any antibody which competes with antibody BOIIB2 for the binding to FVIII.

In other embodiments, affinity ligands of non-immunoglobulin origin are employed as binding ligands instead of, or together with, immunoglobulins.

The diversity needed for selection of affinity ligands may be generated by combinatorial engineering of one of a plurality of possible scaffold molecules, and specific and/or selective affinity ligands are then selected using a suitable selection platform. The scaffold molecule may be of immunoglobulin protein origin (Bradbury A R and Marks J D (2004) J. Immunol. Meths . 290:29-49), of non-immunoglobulin protein origin (Nygren P and Skerra A (2004) J. Immunol. Meths. 290:3-28), or of an oligonucleotide origin (Gold L et al. (1995) Annu . Rev. Biochem. 64:763-797).

A large number of non-immunoglobulin protein scaffolds have been used as supporting structures in development of novel binding proteins. Non-limiting examples of such structures, useful for generating affinity ligands for use according to the present disclosure, are staphylococcal protein A and domains thereof and derivatives of these domains, such as protein Z (Nord K et al. (1997) Nat. Biotechnol. 15:772- 777); lipocalins (Beste G et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96:1898-1903); ankyrin repeat domains (Binz H K et al. (2003) J. Mol. Biol. 332:489-503); cellulose binding domains (CBD) (Smith G P et al . (1998) J. Mol. Biol. 277:317-332; Lehtio J et al. (2000) Proteins 41:316-322); gamma crystallines (Fiedler U and Rudolph R, WO01/04144); green fluorescent protein (GFP) (Peelle B et al. (2001) Chem. Biol. 8:521-534); human cytotoxic T lymphocyte- associated antigen 4 (CTLA-4) (Hufton S E et al. (2000) FEBS Lett. 475:225-231; Irving R A et al . (2001) J. Immunol. Meth. 248:31-45); protease inhibitors, such as Knottin proteins (Wentzel A et al. (2001) J. Bacteriol. 183:7273- 7284; Baggio R et al. (2002) J. Mol. Recognit. 15:126-134) and Kunitz domains (Roberts B L et al. (1992) Gene 121:9-15; Dennis M S and Lazarus R A (1994) J. Biol. Chem. 269:22137- 22144); PDZ domains (Schneider S et al . (1999) Nat. Biotechnol. 17:170-175); peptide aptamers, such as thioredoxin (Lu Z et al. (1995) Biotechnology 13:366-372; Klevenz B et al . (2002) Cell. Mol. Life. Sci. 59:1993-1998); staphylococcal nuclease (Norman T C et al. (1999) Science 285:591-595); tendamistats (McConell S J and Hoess R H (1995) J. Mol. Biol. 250:460-479; Li R et al. (2003) Protein Eng. 16:65-72); trinectins based on the fibronectin type III domain (Koide A et al. (1998) J. Mol. Biol. 284:1141-1151; Xu L et al. (2002) Chem. Biol. 9:933-942); and zinc fingers (Bianchi E et al. (1995) J. Mol. Biol. 247:154-160; Klug A (1999) J. Mol. Biol. 293:215-218; Segal D J et al. (2003) Biochemistry 42:2137-2148).

The above mentioned examples of non-immunoglobulin protein scaffolds include scaffold proteins presenting a single randomized loop used for the generation of novel binding specificities, protein scaffolds with a rigid secondary structure where side chains protruding from the protein surface are randomized for the generation of novel binding specificities, and scaffolds exhibiting a non-contiguous hyper-variable loop region used for the generation of novel binding specificities.

In addition to non-immunoglobulin proteins, in some embodiments, oligonucleotides may also be used as affinity ligands. Single stranded nucleic acids, called aptamers or decoys, fold into well-defined three-dimensional structures and bind to their target with high affinity and specificity.

(Ellington A D and Szostak J W (1990) Nature 346:818-822; Brody E N and Gold L (2000) J. Biotechnol. 74:5-13; Mayer G and Jenne A (2004) BioDrugs 18:351-359) . The oligonucleotide ligands can be either RNA or DNA.

For selection of the desired affinity ligand from a pool of variants of any of the scaffold structures disclosed herein, a number of selection platforms are available for the isolation of a specific novel ligand against the target FVIII. Selection platforms include, but are not limited to, phage display (Smith G P (1985) Science 228:1315-1317), ribosome display (Hanes J and Pluckthun A (1997) Proc. Natl. Acad. Sci. U.S.A. 94:4937-4942), yeast two-hybrid system (Fields S and Song 0 (1989) Nature 340:245-246), yeast display (Gai S A and Wittrup K D (2007) Curr Opin Struct Biol 17:467-473), mRNA display (Roberts R W and Szostak J W (1997) Proc. Natl. Acad. Sci. U.S.A. 94:12297-12302), bacterial display (Daugherty P S (2007) Curr Opin Struct Biol 17:474-480, Kronqvist N et al. (2008) Protein Eng Des Sel 1-9, Harvey B R et al. (2004) PNAS 101 (25) : 913-9198) , microbead display (Nord 0 et al. (2003) J Biotechnol 106:1- 13, WO01/05808), SELEX (System Evolution of Ligands by Exponential Enrichment) (Tuerk C and Gold L (1990) Science 249:505-510) and protein fragment complementation assays (PCA) (Remy I and Michnick S W (1999) Proc. Natl. Acad. Sci. U.S.A. 96 : 5394-5399) .

Matrix

In some embodiments, an affinity matrix is provided comprising a polymeric support that is modified with the affinity ligand. The polymeric support is generally any polymer capable of being used as an affinity matrix. The polymer preferably exhibits low non-specific protein binding and high binding capacity for the affinity ligand with which it may be modified. The polymers can be natural or synthetic. The natural or synthetic polymers may be cross- linked. Such polymers include but are not limited to natural agarose, cross-linked agarose, dextran, cross-linked dextran, xylan alginate, chitosan, beaded cellulose, polyacrylamide , polyacrylate , polystyrene, polymethacrylate , polycaprolactones , polyoxyethylenes , polyvinyl resins, agarose-polyacrylamide copolymers, and dextran- polyacrylamide copolymers. Combinations of these and other polymers are also included within the scope of the invention, provided that the combined polymers possess the desired characteristics of low non-specific protein binding and high binding capacity for the affinity ligand. In some embodiments, polymers include those employed in affinity based molecular pull down and/or immunoprecipitat ion methods .

Natural agarose, cross-linked agarose, polyacrylamide, and cross-linked dextran are the preferred polymers for use in the invention.

Cross-linked agarose is the more preferred polymer for use in the invention.

The polymeric support can be soluble or insoluble in aqueous solutions. Preferably, the polymeric support is insoluble in aqueous solutions. In some embodiments, the polymeric support is in the form of particles or beads.

In one embodiment, the affinity matrix comprises a particulate polymeric support in which the particles have an average size of less than about 1,000 micrometers. Typically, the particles in the particulate polymeric support will have an average size of less than about 700 microns, more typically less than 600 microns. For example, for some applications the particles of the particulate polymeric support will have an average size less than about 400 microns and in other applications the particles will have an average size less than about 200 microns.

The particles of natural and cross-linked agarose typically range from about 20 to about 300 micrometers in diameter. Preferably, the particles of natural and cross-linked agarose range from about 30 to about 250 micrometers in diameter. More preferably, the particles of natural and cross-linked agarose range from about 40 to about 165 micrometers in diameter.

The particles of polyacrylamide typically range from about 20 to about 200 micrometers in diameter. Preferably, the particles of polyacrylamide range from about 30 to about 150 micrometers in diameter. More preferably, the particles of polyacrylamide range from about 40 to about 105 micrometers in diameter.

The particles of dextran and cross-linked dextran typically range from about 15 to about 600 micrometers in diameter. Preferably, the particles of dextran and cross-linked dextran range from about 20 to about 400 micrometers in diameter. More preferably, the particles of dextran and cross-linked dextran range from about 50 to about 300 micrometers in diameter.

The polymeric support can be modified with the affinity ligand. For example, anti-FVIII antibodies, or antigen binding fragments thereof, may be linked to the polymeric support such as the agarose and cross-linked agarose derivatives sold under the trademarks SEPHAROSE 6B, CL6B, 4B and CL4B by Pharmacia Fine Chemicals or those sold under the trademarks Bio-Gel A-0.5M, A-1.5M, and A-50M by Bio-Rad Laboratories of Richmond Calif; or, for example, cross- linked dextran sold under the trademark SEPHADEX, and polyacrylamide beads sold under the trademarks Bio-Gel P-2, P-30, P-100 and P-300 also by Bio-Rad Laboratories are also useful solid matrixes. In some embodiments, an agarose matrix is activated for linking using cyanogen bromide. The activated matrix may then be washed and linked to the desired affinity ligand (e.g., anti-FVIII antibody molecules) without drying of the activated matrix. The matrix-linked affinity ligand may then be washed and then ready for use. Unreacted reactive groups on the matrix, if any, optionally, can be reacted with e.g. an amine such as ethanolamine or Tris.

In some embodiments, the affinity matrix may be used in its loose state, as in a beaker or flask, or it may be confined in a column. In other embodiments, prior to use, it is preferable that the affinity matrix be washed in the buffer or other aqueous medium utilized for purification of cell lysates containing the FVIII to eliminate non-specifically bound proteins or those antibodies that were unstably linked to the support .

For example, in some embodiments, an aqueous composition comprising FVIII (e.g., an expression vector-transfected cell lysate expressing FVIII) is provided, and is then admixed with the affinity matrix comprising the polymeric support that is modified with the affinity ligand. That admixture forms a reversible, linked ant ibody-F I I I complex between the linked antibody and the FVIII. The antibody- FVIII complex is then separated from the remainder of the un-complexed aqueous composition to thereby obtain the FVIII in purified form linked to the affinity matrix. When the admixture takes place in a beaker or flask, this separation can be made by filtration and washing. When the matrix is in a column, the separation may take place by elution of the uncomplexed aqueous medium, again, preferably, followed by a washing step.

When the purified FVIII is desired free from the affinity matrix, it can typically be obtained by a variety of one or more steps. For example, the reversible linked antibody- FVIII complex is dissociated into its component parts of matrix-linked antibody and FVIII, followed by separating the FVIII from the linked antibody to provide a solution of the purified FVIII free from the affinity matrix. The solution may be used as such, or it may be concentrated or dried prior to use as desired. In some embodiments, it may be desirable to desalt and/or sterilize the solution prior to use, as is known to one of ordinary skill in the art.

In one embodiment, the reversible complex is dissociated using a glycine hydrochloride (glycine-HCl) solution (e.g., about 0.1 molar (M) to about 0.2 M at an acidic pH (e.g., about 2.5 to about 2.7) . In another embodiment, the bound FVIII is competed away from the matrix-linked antibody by admixture of the reversible complex with an excess of the immunogenic molecule utilized to raise the FVIII antibodies.

Substances that reduce the polarity of a buffer may facilitate elution without affecting protein activity: e.g., dioxane (up to about 10%) ; ethylene glycol (up to about 50%) .

SPACERS

One of ordinary skill in the art knows that for the coupling of an antibody to a solid matrix to have maximum affinity for the antigen it is important that the ligand retain its active conformation after coupling to the matrix. Antibody molecules exist in their active forms only in a small number of conformations and the functional affinities can vary widely upon coupling to a solid surface. By increasing the distance between the substrate and the active binding site, for example by the use of a "spacer" or "spacer arm," additional binding capacity may be possible.

Accordingly, in some embodiments, increased antibody activity is possible by the introduction of a hydrophilic spacer arm. For example, an intermediate compound or spacer can be attached to the solid phase and the antibody affinity matrix can then be immobilized on the solid phase by attaching the affinity matrix to the spacer. The spacer can itself be a ligand (i.e., a second ligand) that has a specific binding affinity for the free antibody affinity matrix .

In one embodiment, the spacer is poly-L-lysine . For example, a method may involve a polycarbonate surface coated with poly-L-lysine (Sigma, Cat. #P6516) . The amino group at one end of the poly-L-lysine, a 14.6 kD hydrophilic spacer consisting of approximately 113 lysine monomers, activates the carbonate group of polycarbonate membrane matrix. The amino group at the other end of the poly-L-lysine is activated by addition of glutaraldehyde . The glutaraldehyde also reacts with unprotected sites on the membrane's surface. The protein links to glutaraldehyde cross-linked to the membrane matrix or to the poly-L-lysine.

In other embodiments, the spacer is polyethylene glycol PEG.

In some embodiments, the present invention provides a method for preparing a FVIII, the method comprising contacting, in the presence of at least about 300 mM CaCl2, a composition comprising the FVIII with an anti-FVIII antibody attached to a matrix via a hydrophilic spacer arm, wherein the antibody specifically binds the FVIII, wherein the FVIII does not bind to any vWF that may be present in the composition.

In one embodiment, the anti-FVIII antibody is a single heavy chain antibody.

In another embodiment, the present invention provides a method for preparing a FVIII, the method comprising contacting, in the presence of at least about 300 mM CaCl2, a composition comprising the FVIII with the affinity resin VlllSelect, wherein the FVIII binds to the VlllSelect, but not to any vWF that may be present in the composition. VlllSelect is an affinity medium designed for the purification of recombinant BDD-FVIII. This affinity media uses BAC's CaptureSelect® Factor VIII ligand. The VlllSelect affinity resin is obtained from GE Healthcare (Piscataway, NJ) (Catalog No. 17-5450) .

In other embodiments, the method for purifying the FVIII further comprises washing the affinity ligand with a wash buffer solution under a condition sufficient to maintain the complex between the FVIII and the affinity ligand.

In some embodiments, the method further comprises eluting the FVIII from the affinity ligand with an elution buffer.

In one embodiment, the wash buffer comprises about 300 mM to about 1000 mM NaCl.

The following examples are given only to illustrate the present process and are not given to limit the invention. One skilled in the art will appreciate that the examples given only illustrate that which is claimed and that the present invention is only limited in scope by the appended claims . EXAMPLES Example 1 FVIII Purification

Recombinant or plasma derived FVIII/vWF complexes comprising Factor VIII (e.g., full length and/or BDD-FVIII) and vWF (e.g., truncated and/or modified vWF) are removed from the growth media or formulation buffer to obtain a composition comprising the complexes prior to loading onto the Factor VIII affinity column (e.g., VlllSelect column) . The composition is diluted, resuspended, and/or buffer exchanged into a buffer (e.g., Load/Equilibration buffer: 10 mM histidine, 20 mM calcium chloride, 300 mM sodium chloride, 0.02% Tween 80 at pH 7.0) suitable for the affinity matrix. Depending on how the buffer exchange is performed, a final calcium chloride concentration of 300-400 mM is provided to separate the FVIII from the vWF . The Factor VIII affinity column is equilibrated with the Load/Equilibration buffer supplemented with 300-400 mM CaCl₂. The sample is loaded on to the column and the column is washed in an appropriate wash buffer (e.g., Wash buffer 1: 20 mM histidine, 20 mM calcium chloride, 300 mM sodium chloride, and 0.02% Tween 80 at pH 6.5, Wash buffer 2: 20 mM histidine, 20 mM calcium chloride, 1.0 M sodium chloride, and 0.02% Tween 80 at pH 6.5, etc.). The FVIII is then eluted from the affinity resin. An example buffer for Vlllselect would be, for example: Elution buffer: 20 mM histidine, 20 mM calcium chloride, 2.0 M sodium chloride, and 0.02% Tween 80 dissolved in 70% ethylene glycol at pH 6.5.

The sample then is buffer exchanged into a matrix suitable for FVIII (e.g., Formulation buffer: 1% Human Serum Albumin, 0.3% Sucrose, 2.2% Glycine, 0.02M Histidine, 0.22M NaCl, 0.0025M CaC12, 0.008% Tween 80 pH 7.0).

Example 2

Purification of Plasma-derived FVIII

Two aliquots of 10 mL of a pre-purified, plasma-derived FVIII/vWF complex (at78U FVIII/mL) were diluted to 50 mL with 40 mL of Load/Equilibration buffer (10 mM histidine, 20 mM calcium chloride, 300 mM sodium chloride, 0.02% Tween 80 at pH 7.0) . One sample contained an additional ~400 mM of CaCl₂ whereas the other sample had no additional calcium added to it . Initial conditions for VlllSelect (GE Healthcare, Piscataway, NJ) (Catalog No. 17-5450) column were identical to those provided by GE in the product information file. A 1 cm (h) , XK16 column was packed at 200 cm/hr. Depending on the sample being run, the column was equilibrated with Load/Equilibration buffer ± 400mM CaCl₂. The column flow rate was maintained at 167 cm/hr.

Following loading of the sample the column was washed for 5 CV with load/equilibrium buffer, 5 CV of wash buffer 1, and 5 CV of wash buffer 2 and then eluted with the elution buffer (1CV) .

The resin was cleaned (in reverse flow) at 0.01 M NaOH, 2.0 M NaCl and held for NMT 1 hr before re-equilibration into the load buffer.

The samples were then assayed for presence of FVIII activity by a chromogenic assay: Load = 15.6 U/mL (50mL total); Elution (no additional CaCl₂) = 4.7U/mL (4mL total); and Elution (400mM additional CaCl₂) = 43.lU/mL (4mL total). Recovery in the presence of CaCl2 ~10x compared to loading in the absence of CalCl₂. Figure 1 is 7% Tris-Acetate gel showing the purification process.

Example 3

Purification of Recombinant FVIII

The ideal conditions for precipitation of a recombinant BDD- FVIII from a recombinant BDD-F I I I /vWF complex from the growth media was a 1/1 addition of 50% PEG 600, 0.25 M Tris, pH 7.0 and incubated on ice for 1 hour. The precipitated pellet was collected by centrifugat ion . The pellet was re- suspended (initially) in 100 mL of the VlllSelect Load/Equilibration buffer (10 mM histidine, 20 mM calcium chloride, 300 mM sodium chloride, 0.02% Tween 80 at pH 7.0) (~2/5^th initial sample volume) and filtered to remove gross solids with a 40 μπι nylon membrane. An additional 150 mL of the Load/Equilibration buffer was used to wash the filter, followed by 28 mL of 5 M Ca²⁺ (collected separately). A 0.45 + 0.22 μπι Sartobran P Sterile MidiCap filter was used to remove any remaining particles from the sample. The filter was then washed with the 5M Ca²⁺ and an additional 72 mL of Load/Equilibrium buffer.

Initial conditions for VlllSelect column were identical to those provided by (GE Healthcare, Piscataway, NJ) (Catalog No. 17-5450) in the product information file. A 1 cm (h) , XK16 column was packed at 200 cm/hr. The column flow rate was maintained at 167 cm/hr. Following loading of the sample, the column was washed for 5 CV with Load/Equilibrium buffer, 5 CV of wash buffer 1, and 5 CV of wash buffer 2 and then eluted with the elution buffer. The resin was cleaned (in reverse flow) at 0.01 M NaOH, 2.0 M NaCl and held for NMT 1 hr before re-equilibration into the load buffer.

The VlllSelect eluate was exchanged into the Factor VIII Storage Buffer using a HiPrep 26/10 Desalting column from GE Healthcare (Piscataway, NJ) .

The result are shown in Figure 2, which is a 7% Tris-Acetate gel showing the purification process: Lane 1: ~10 I.U. Xyntha® Antihemophilic Factor ( recombinantly produced in non-human cell lines) (Wyeth Pharmaceuticals Inc., Philadelphia, PA); Lane 2: Supernatant; Lane 3: Resuspended PEG pellet; Lane 4 Filtered Load; and Lane 5: Flow through.

Claims

1. A method for preparing a Factor FVIII (FVIII), the method comprising:

contacting the FVIII with an affinity ligand in the presence of an amount of a doubly charged ion, wherein the amount of the doubly charged ion is sufficient for the FVIII to form a complex with the affinity ligand but not a vWF .

2. The method of claim 1, wherein the affinity ligand is an antibody that specifically binds the FVIII.

3. The method of claim 2, wherein the antibody is a single heavy chain antibody.

4. The method of claim 1, wherein the step of contacting comprises :

contacting the affinity ligand with a composition comprising (i) the FVIII; (ii) the vWF; and (ii) the amount of the doubly charged ion.

5. The method of claim 1, wherein the step of contacting comprises :

(b) providing the amount of the doubly charged ion.

6. The method of claim 1, wherein the step of contacting comprises : (a) equilibrating the affinity ligand with the amount of the doubly charged ion; and

7. The method of claim 1, wherein the affinity ligand is attached to a matrix.

8. The method of claim 1, wherein the affinity ligand is attached to a matrix via a hydrophilic spacer arm.

9. The method of claim 1, wherein the amount of the doubly charged ion is at least about 300 mM.

10. The method of claim 1, wherein the doubly charged ion is a calcium ion.

11. The method of claim 1, wherein the doubly charged ion

12. The method of claim 1 further comprising:

washing the affinity ligand with a wash buffer solution under a condition sufficient to maintain the complex between the FVIII and the affinity ligand.

13. The method of claim 12, wherein the wash buffer comprises about 300 mM to about 1000 mM NaCl .

14. The method of claim 12 further comprising: eluting the FVIII from the affinity ligand with an elution buffer .

15. The method of claim 14, wherein the elution buffer comprises at least about 1500 mM NaCl.

16. The method of claim 1 further comprising recombinantly co-expressing the FVIII with the vWF prior to the step of binding .

17. A composition comprising a FVIII, wherein the FVIII is prepared according to the method of claim 1.

18. A composition comprising a FVIII bound to an affinity ligand in the presence of an amount of a doubly charged ion, wherein the amount is sufficient to prevent binding of a vWF to the FVIII.

19. An affinity ligand equilibrated with an amount of a doubly charged ion and having a FVIII bound thereto, wherein the amount of the doubly charged ion is sufficient for the FVIII to form a complex with the affinity ligand but not a vWF .