WO2013093760A2

WO2013093760A2 - Compositions, methods, and kits for preparing sialylated recombinant proteins

Info

Publication number: WO2013093760A2
Application number: PCT/IB2012/057392
Authority: WO
Inventors: Thomas Barnett
Original assignee: Grifols, S.A.
Priority date: 2011-12-19
Filing date: 2012-12-17
Publication date: 2013-06-27
Also published as: UY34527A; AU2012340501A1; WO2013093760A3; AR089324A1

Abstract

A method for altering a sialylation pattern of one or more recombinant proteins expressed in a cell, wherein the cell is genetically engineered to recombinantly express an α2,3-sialyltransferase to sialylate the protein. This expression system increases the sialylation of the protein that is expressed, in particular the amount of an α-2,3-linked sialic acid attached to the protein.

Description

COMPOSITIONS, METHODS, AND KITS FOR PREPARING SIALYLATED RECOMBINANT PROTEINS FIELD OF THE INVENTION

The present invention involves altering the sialylation capabilities of a cell to effect sialylation of one or more recombinant proteins expressed therein. BACKGROUND

Under-representation of sialic acid has been shown to be highly correlated with reduced persistence of certain recombinant proteins in plasma. As a consequence, glycosylated proteins with reduced sialic acid content often exhibit poor pharmacokinetics in animal models that is unacceptable. Several in vitro methods including chemical methods have been proposed for enhancing the sialylation of proteins, but such in vitro methods are typically inefficient and expensive, and can lead to a heterogeneous population of molecular forms. For example, significant chemistry can be involved in attaching sialic acid to proteins in vitro, and this is rendered all the more difficult especially when large molecules such as, for example, FVIII (FVIII) (a/k/a "anti-hemophilic factor" (AHF) ) or von Willebrand factor (vWF) are considered.

An effective method for directing, and more efficiently attaching, sialic acid to proteins is needed, Also needed are proteins having increased sialylation, in part icular recombinantly prepared therapeutic proteins, and methods, compositions, and kits related thereto.

BRIEF DESCRIPTION OF THE FIGURE

Figure 1 is: (A) a schematic illustration of one embodiment depicting a mammalian cell transfected with a B-domain-deleted FVIII plasmid carrying the neomycin/G418 resistance gene into which a second different expression plasmid has been further transfected, coding for a2 , 3-sialyltransferase and truncated variants of von Willebrand factor, and carrying zeocin resistance; and (B) a schematic illustration of one embodiment depicting a mammalian cell transfected with a B-domain-deleted FVIII plasmid carrying the neomycin/G418 resistance gene into which two different expression plasmids have been further transfected, one coding for a2 , 3-sialyltransferase (with zeocin resistance) and one encoding full-length von Willebrand factor (with hygromycin resistance) .

Co-transfection results in linearization of plasmids and incorporation of the DNAs into the cell genome. Other plasmid combinations expressing the three different kinds of human genes described can likewise be constructed.

Figure 2 shows chromatograms of acid released sialic acid of recombinant FVIII proteins analyzed by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) . A. Standard mix showing positions of N-Acetylneuraminic acid (NeuSAc) , Keto (KDN) and N-glycolylneuraminic acid (NeuSGc) ; B. Chromatogram of products of Xyntha® cleavage; C. Chromatogram of products of Kogenate® cleavage; and D. Chromatogram of products of recombinant FVIII cleavage from a clone of the present invention . Figure 3 shows an over-laid chromatogram from the Neu5Gc region of commercial FVIII products and the recombinant FVIII of the present invention.

Figure 4 shows chromatograms for detection and quantitation of Tyr-1680 sulfation by selected ion monitoring (SIM) on a tandem LC-MS/MS system. (A) Both sulfated and non-sulfated Tyrl680 are detected in Xyntha; (B) Non-sulfated Tyrl680 is not detected in B-domain-deleted FVIII (BDD-FVIII) of recombinant FVIII proteins of the present invention; (C) Non-sulfated Tyrl680 is detected in the same recombinant FVIII sample after forced chemical de-sulfation by acid (intended as a positive control) .

Figure 5 shows a fitted curve of the binding of recombinant FVIII proteins (prepared from different cell clones of PER.C6 transfectants that express recombinant BDD-FVIII) to antibody-bound plasma-derived vWF .

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method for preparing a recombinant protein, the method comprising: co-expressing the recombinant protein with a recombinant a2 , 3-sialyltransferase in a mammalian cell under a condition sufficient to increase sialylation of the recombinant protein in the cell. In another aspect, the present invention provides a method for preparing a recombinant FVIII protein, the method comprising: co-expressing the recombinant FVIII protein with a recombinant a2 , 3-sialyltransferase in a mammalian cell under a condition sufficient to increase sialylation of the recombinant protein in the cell, wherein the cell further expresses a recombinant vWF protein.

In other aspects, the present invention provides a method for preparing a recombinant FVIII protein, the method comprising: co-expressing the recombinant FVIII protein with a recombinant a2 , 3-sialyltransferase in a mammalian cell under a condition sufficient to increase sialylation of the recombinant protein in the cell, wherein the cell further expresses a recombinant vWF protein lacking a propeptide, wherein the cell further expresses the propeptide.

In some aspects, recombinant FVIII and/or vWF proteins prepared by the methods of the present invention are provided .

In still further aspects, pharmaceutical compositions comprising the recombinant ly expressed proteins of the present invention are provided.

In another aspect, cells and cell cultures are provided as described herein. In other aspects, methods for treating a blood disorder in a mammal are provided, the method comprising administering the compositions described herein comprising the recombinant proteins .

In still further aspects, the present invention provides kits comprising expression vectors and reagents for recombinantly expressing the proteins of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In one aspect, the present invention provides a method for preparing a recombinant protein. The method comprises co-expressing the recombinant protein with a recombinant a2 , 3-sialyltransferase in a mammalian cell under a condition sufficient to increase sialylation of the recombinant protein in the cell.

In some embodiments, following recombinant expression of the protein by the cell, the recombinant protein has increased level of sialylation compared to the same protein that is expressed in cells that do not co-express the recombinant a2 , 3-sialyltransferase . The sialic acid content of glycoproteins can be determined by a variety of methods, for example as described by U.S. Patent Application Publication No. 2011/0086362 to Wang et al.; and Reuter & Schauer, Methods Enzymol . , 230:168-99 (1994), which are herein incorporated by reference for their teaching of determining sialic acids. By way of another example, Prozyme's Rapid Sialic Acid Quatitation Kit (Product Code GS300, Prozyme®, San Leandro, CA) provides a method for sialic acid quantitation based on enzymatic release of sialic acid from a sialylated protein, then converting released sialic acid to hydrogen peroxide, which reacts with a dye stoichiometrically, generating fluorescence.

In preferred embodiments, the recombinant a2 , 3-sialyltransferase is a mammalian enzyme, more preferably a human enzyme.

In other embodiments, a nucleic acid encoding the a2 , 3-sialyltransferase in expressible format is transiently or stably introduced into cells, and the a2 , 3-sialyltransferase is expressed during the culturing of the cells according to the invention when the recombinant protein (s) of interest also is/are co-expressed. This results in an altered sialylation pattern of the recombinant protein (s) of interest as compared to when no recombinant a2 , 3-sialyltransferase is expressed in the cells.

Nucleic acids encoding the protein of interest or a2 , 3-sialyltransferase preferably are each under control of a heterologous promoter, which should be active or have the possibility of being regulated in the cells of the invention. In some embodiments, the encoding nucleic acids are each integrated into the genome of the cells, to ensure stable inheritance, and provide for stable expression in subsequent generations of the cells. Recombinant expression of a2 , 3-sialyltransferase is disclosed by, e.g., U.S. Patent No. 7,642,078, which is herein incorporated by reference for its teaching of recombinant expression of a2 , 3-sialyltransferase . Examples of nucleotide and amino acid sequences for an a2 , 3-sialyltransferase include, without limitation, the sequences as set forth in SEQ ID NOs : 1 and 2.

Nucleotide and amino acid sequences for an a2 , 3-sialyltransferase also is disclosed by NCBI Reference Sequence: NM_006278.1 , which is herein incorporated by reference for its teaching of a2 , 3-sialyltransferase sequences . In some embodiments, the recombinant protein that is co-expressed with the recombinant a2 , 3-sialyltransferase comprises a primary, secondary, tertiary, and/or higher order structure that renders the protein amenable to sialylation by the recombinant a2 , 3-sialyltransferase . Preferably, the primary amino acid sequence of the recombinant protein comprises one or more N-glycosylation sites. N-glycosylation sites may be native to the protein, or a nucleic acid sequence may be genetically engineered/altered to encode a protein having such a site (s) .

An "N-glycosylation site" has the sequence N-X-S/T/C, wherein X is any amino acid residue except proline, N is asparagine and S/T/C is serine, threonine or cysteine, preferably serine or threonine, and most preferably threonine .

Recombinant proteins of human and non-human (e.g., primates, dogs, cats, horses, pigs, mice, rats, guinea pigs, rabbits, cows, other vertebrates) origin are within the scope of the present invention. Also within the scope of the present invention are proteins corresponding to wild-type proteins or variants thereof.

The term "variant" as used herein in the context of a protein is defined as a molecule in which the amino acid sequence of a naturally occurring molecule has been modified (i.e., by way of modification of the encoding nucleotide sequence) and is intended to include mutants, truncations, chimeric proteins, etc. Variants falling within this invention may possess amino acid substitutions, deletions, and/or insertions. Amino acid substitutions in a particular protein may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and/or the amphipathic nature of the residues involved. Also included within the definition of variant are those proteins having additional amino acids at one or more sites of the C-terminal, N-terminal, and within the sequence.

Accordingly, in some embodiments, the proteins of the invention including the recombinant therapeutic proteins that are sialylated and the recombinant a2 , 3-sialyltransferases include the naturally occurring forms as well as variants thereof. In other embodiments, the variants are substantially homologous and/or functionally equivalent to the native protein on which they are based. As used herein, two proteins (or a region of the proteins) are "substantially homologous" when the amino acid sequences are at least about 75%, preferably at least about 75%, 80%, 85%, 90%, 95%, 98% or more identical. In one embodiment, a variant differs by 1, 2, 3, 4, or more amino acids. A variant polypeptide, in other embodiments, can differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, and truncations or a combination of any of these. By "functionally equivalent" is intended that the sequence of the variant provides a protein having substantially the same biological activity as the native protein or at least one of the functions typically identified as part of the biological activity of the molecule. Such functionally equivalent variants that comprise substantial sequence variations are also encompassed by the invention. Thus, a functionally equivalent variant of the native protein will have a sufficient biological activity to be functionally or therapeutically useful.

Methods are available in the art for determining functional equivalence. Biological activity can be measured using assays specifically designed for measuring activity of the native protein. Additionally, antibodies raised against the biologically active native protein can be tested for their ability to bind to the functionally equivalent variant, where effective binding is indicative of a protein having conformation similar to that of the native protein. In some embodiments, variant polypeptides can be fully functional or can lack function in one or more activities. Thus, in the present case, variations can affect the function, for example, of one or more of the modules, domains, or functional sub-regions of the protein molecules of the present invention.

Fully functional variants typically contain only conservative variation or variation in non-critical residues or in non-critical regions. Functional variants can also contain substitution of similar amino acids, which result in no change or an insignificant change in function. Alternatively, such substitutions may positively or negatively affect function to some degree. Amino acids that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al . , Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity. Sites that are critical can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling. Orthologs, homologs, and allelic variants can be identified using methods well known in the art.

For example, to determine the percent homology of two amino acid sequences, or of two nucleotide sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of one protein or nucleic acid for optimal alignment with the other protein or nucleic acid) . The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in one sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the other sequence, then the molecules are homologous at that position. As used herein, amino acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid "identity". The percent homology between the two sequences is a function of the number of identical positions shared by the sequences (i.e., percent homology equals the number of identical positions/total number of positions times 100) .

In some embodiments, the present invention provides proteins or polypeptides having a lower degree of identity but having sufficient similarity so as to perform one or more of the same functions performed by the proteins described herein. Similarity is determined by conserved amino acid substitution. Such substitutions are those that substitute the given amino acid in a polypeptide by another amino acid of like characteristics. Conservative substitutions are likely to be phenotypically silent. Guidance concerning which amino acid changes are likely to be phenotypically silent are found in Bowie et al., Science 247:1306-1310 (1990) . Both identity and similarity can be readily calculated. Preferred computer program methods to determine identify and similarity between two sequences include, but are not limited to, BLASTP, BLASTN, and FASTA (Atschul, S. F., J. Molec. Biol. 215:403 (1990)); utilizing the default parameters available within the programs.

GenBank Accession Nos . : X01683 ; NP_000123, M14113; ABV90867.1; and NM_000552; each of which is herein incorporated by reference in its entirety, disclose amino acid and nucleic acid sequences of one or more proteins that can be recombinantly co-expressed with the recombinant a2 , 3-sialyltransferase in accordance with the present invention . In some embodiments, the recombinant protein that is co-expressed with the recombinant a2 , 3-sialyltransferase is a FVIII (e.g., full length FVIII, B domain-deleted FVIII) .

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, variants of FVIII, as described in, U.S. Pat. No. 5,668,108 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 .

In some embodiments, the recombinant protein that is co-expressed with the recombinant a2 , 3-sialyltransferase comprises an amino acid sequence as set forth in SEQ ID NO: 3, 4, 5, or 6.

In other embodiments, the recombinant protein that is co-expressed with the a2 , 3-sialyltransferase is a recombinant vWF protein. In one embodiment, the recombinant vWF protein comprises the amino acid sequence as set forth in SEQ ID NO : 7 , 8, 9, or 10.

In other embodiments, the recombinant protein that is co-expressed with the recombinant a2 , 3-sialyltransferase is a vWF variant that comprises a first amino acid sequence contiguous with a second amino acid sequence, wherein the first sequence corresponds to at least a portion of the vWF protein, wherein the second sequence corresponds to an antibody Fc fragment. In one embodiment, the recombinant protein that is co-expressed with the a2 , 3-sialyltransferase is a recombinant vWF polypeptide having a first amino acid sequence as present in a vWF polypeptide and a second amino acid sequence heterologous to the first, wherein the polypeptide is capable of binding a FVIII.

As used herein, the term "capable of binding" contemplates embodiments wherein the capability of the recombinant vWF polypeptide to bind to a FVIII is effected by higher order protein assembly and/or one or more post-translational modifications such as, for example, signal peptide cleavage, propeptide cleavage, propeptide association, phosphorylation, glycosylation, and such like. For example, in some embodiments, the recombinant vWF polypeptide is "capable of binding" to FVIII as a dimer, trimer, tetramer, or higher order multimeric complex that forms subsequent to multimerization of the polypeptide. Or, for example, in other embodiments, the recombinant vWF polypeptide is "capable of binding" FVIII following multimerization of the recombinant vWF polypeptide subsequent to association of a propeptide with the recombinant vWF polypeptide. "Multimerization" and "oligomerization" are used interchangeably herein and refer to the association of two or more protein molecules, mediated by covalent (e.g., intermolecular disulfide bonds) and/or non-covalent interactions. Accordingly, "multimer ( s ) " and "oligomer ( s ) " also are used interchangeably herein.

In one embodiment, the first amino acid sequence of the recombinant vWF polypeptide defines a structure or domain that reacts with a monoclonal anti-vWF antibody capable of specifically binding to a region of a reference vWF polypeptide comprising a FVIII binding domain. In one embodiment, the monoclonal antibody is monoclonal antibody C3 as described by, e.g., Foster et al., JBC, 262:8443 (1987) and Fulcher et al., J. Clin. Invest., 76:117 (1985), each of which is herein incorporated by reference for its teaching of monoclonal antibody C3 and method of preparing monoclonal antibodies, in particular monoclonal antibody C3.

Non-limiting examples of vWF amino acid sequences and nucleic acid sequences encoding vWF or a portion thereof are disclosed by, e.g., GenBank Accession Nos . : NP_000543, NM_000552, AAE20723, AAB59512, P04275, EAW88815, ACP57027, EAW88816, and AAD04919; U.S Patent No. 5,260,274; Titani et al . , Biochemistry, 25:3171-84 (1986); and Sadler et al., PNAS, 82:6391-6398 (1985), each of which is herein incorporated by reference for its teaching of amino acid and nucleic acid sequences corresponding to vWF .

A person of ordinary skill in the art knows that the prototypical preprop-vWF is a polypeptide of 2813 amino acids with a signal peptide of 22 amino acids and repetitive functional domains, A, B, C, D and CK, which are distributed from the amino terminal in the order "Dl", "D2", "D"', "D3", "Al", "A2", "A3", "D4", "Bl", "B2", "B3" (the latter three collectively considered "B"), "CI", "C2", and "CK". The "mature" vWF subunit is composed of, from the N- to the C-terminus in the order, the domains: D' -D3-A1-A2-A3-D4-B1-B2-B3-C1-C2-CK.

An amino acid sequence of an exemplary full-length human vWF is shown by SEQ ID NO : 8 , which is encoded by nucleotides 251-8689 of SEQ ID NO:ll. With reference to SEQ ID NO:8, the "signal peptide" portion of vWF spans amino acid positions 1 through Cys-22, the "propeptide" portion (D1-D2) spans amino acid positions 23 through Arg-763, and the "mature" vWF protein spans amino acid positions 764 through 2813. The individual domains have also been approximately mapped as D' : 764 - 865; D3: 866 - 1242; Al : 1260 - 1479; A2 : 1480 - 1672; A3: 1673 - 1874; D4 : 1947 - 2298; B: 2296 - 2399; CI: 2400 - 2516; C2 : 2544 - 2663; and CK: 2720 - 2813. An alternative vWF domain mapping and naming system has been used by the EXPASY Protein Database convention (worldwideweb.uniprot.org/uniprot/P04275) as Dl: 34 - 240; D2 : 387 - 598; D' : 776 - 827; D3: 866 - 1074; Al : 1277 - 1453; A2: 1498 - 1665; A3: 1691 - 1871; D4: 1949 - 2153; B: 2255 - 2328 (which is named CI in EXPASY); CI: 2429 - 2495 (named C2 in EXPASY); C2 : 2580 - 2645 (named C3 in EXPASY); and CK: 2724 - 2812.

One of ordinary skill in the art knows that the ability of the recombinant vWF polypeptide to bind FVIII may be determined in a variety of ways. In particular, the recombinant vWF polypeptide of the present invention may be assayed for its ability to bind the FVIII using techniques described herein and/or adapting techniques known in the art. For example, to analyze/determine binding, immunoassays can be employed including, but are not limited to, competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, etc. (see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York, which is herein incorporated by reference in its entirety) .

For example, the recombinant vWF polypeptide comprising the first and the second amino acid sequences can be contacted with a FVIII in a suitable buffer such as TBS in the presence of a monoclonal antibody coupled to Sepharose. The antibody can be directed against a region of the recombinant vWF polypeptide such that binding of the antibody to the recombinant vWF polypeptide does not interfere with its binding to FVIII (e.g., the antibody may be directed against the second amino acid sequence or an "Al", or "A2" or "A3" repeat region of vWF where such region also is present on the recombinant vWF polypeptide) . Following contact, FVIII bound to the recombinant vWF polypeptide /antibody and unbound FVIII can be separated, e.g. by centrifugat ion, and FVIII can be measured, e.g. using a chromogenic substrate assay (FVIII Coatest; Chromogenix, Molndal, Sweden) .

In preferred embodiments, the first amino acid sequence of the recombinant vWF polypeptide of the present invention is a truncated vWF polypeptide. For example, truncated forms of vWF, in some embodiments, include truncated vWF polypeptides that lack the "Al", "A2", "A3", "D4", "B" (also known as "Bl", "B2", and "B3") , "CI", "C2", and/or "CK" domain of the mature sequence. Other truncated or otherwise modified forms of vWF also are contemplated.

In one embodiment, the first amino acid sequence is as set forth in SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO : 29. In some embodiments, the recombinant protein that is co-expressed with the a2 , 3-sialyltransferase is encoded as a polypeptide consisting of a first amino acid sequence contiguous with a second amino acid sequence, wherein the polypeptide, or a multimer thereof, is capable of binding to a FVIII molecule. In one embodiment, the first amino acid sequence consists of the sequence as set forth in SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO: 26. In other embodiments, the second amino acid sequence of the recombinant vWF polypeptide provides a structure or domain having affinity for a binding partner. The second amino acid sequence is heterologous to the first . In one embodiment, the heterologous second amino acid sequence comprises or consists of a sequence not present in any vWF protein. In one embodiment, at least a portion (e.g., a contiguous portion) of the heterologous second amino acid sequence corresponds to a sequence not present in any vWF polypeptide.

Preferably, in some embodiment, the second amino acid sequence corresponds to an antibody Fc polypeptide such as, e.g., a human IgGl Fc region. For example, the second amino acid sequence can correspond to the amino acid residues that extend from the N-terminal hinge region to the native C-terminus, i.e., is an essentially full-length antibody Fc region. Fragments of Fc regions, e.g., those that are truncated at the C-terminal end, also may be employed. In some embodiments, the fragments preferably contain one or more cysteine residues (at least the cysteine residues in the hinge region) to permit interchain disulfide bonds to form between the Fc polypeptide portions of two separate polypeptides of the present invention, forming dimers.

Other antibody Fc regions may be substituted for the human IgGl Fc region. For example, other suitable Fc regions are those that can bind with affinity to protein A or protein G or other similar Fc-binding matrices, and include the Fc regions of murine IgG, IgA, IgE, IgD, IgM or fragments of the human IgG, IgA, IgE, IgD, IgM Fc region, e.g., fragments comprising at least the hinge region so that interchain disulfide bonds will form.

IgGi Fc region is disclosed by, for example, GenBank Accession no. X70421, which is herein incorporated by reference in its entirety. In one embodiment, the second amino acid sequence comprises the sequence set forth in SEQ ID NO: 30.

In some embodiments, the second amino acid sequence preferably is C-terminus to the first amino acid sequence. Preparation of fusion polypeptides comprising a heterologous amino acid sequence fused to various portions of another amino acid sequence is described, e.g., by Ashkenazi et al., PNAS, 88:10535 (1991) and Byrn et al., Nature 344:677 (1990), each of which is herein incorporated by reference in its entirety. For example, a gene fusion encoding the polypeptide comprising the first and the second amino acid sequences can be inserted into an appropriate expression vector. The expressed fusion proteins can be allowed to assemble, whereby interchain disulfide bonds can form between the polypeptides, yielding dimers . In other embodiments, the fusion polymers of the present invention can be expressed with or without spacer amino acid linking groups. For example, in some embodiments, the recombinant vWF polypeptide of the present invention can further comprise a linker between the first and the second amino acid sequence, wherein the linker comprises one or more amino acid residues separating the first and second sequences . In another embodiment, the recombinant vWF polypeptide of the present invention comprises the amino acid sequence set forth in SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO: 36, or SEQ ID NO: 37. In one embodiment, the polypeptide is encoded by a nucleic acid molecule having the nucleotide sequence set forth in SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, or SEQ ID NO : 46. In other aspects, the present invention provides a recombinant vWF-Fc fusion protein, wherein the vWF portion of the fusion protein is a truncated vWF that lacks at least one domain of a mature full-length vWF polypeptide, wherein the fusion protein is capable of forming multimers that are capable of binding a FVIII protein. In one embodiment, the truncated vWF has domains D' and D3, with the proviso that the truncated vWF lacks domain Al, A2 , A3, D4, Bl, B2, B3, CI, C2, CK, or a combination thereof. In another embodiment, the truncated vWF has domains D' , D3, and Al, with the proviso that the truncated vWF lacks domains A2 , A3, D4, Bl, B2, B3, CI, C2, and CK. In some embodiments, the truncated vWF has domains D' , D3, Al, and A2 , with the proviso that the truncated vWF lacks domains A3, D4, Bl, B2, B3, CI, C2, and CK. In other embodiments, the truncated vWF has domains D' , D3, Al, A2 , and A3, with the proviso that the truncated vWF lacks domains D4, Bl, B2, B3, CI, C2, and CK . In still further embodiments, the truncated vWF lacks domains D4, Bl, B2, B3, CI, C2, and CK. Examples of nucleotide sequences encoding FVIII or vWF include, without limitation, sequences as set forth in SEQ ID NOs:47, 48, and 49.

In other aspects, a nucleotide sequence encoding a recombinant vWF polypeptide of the present is provided, wherein the recombinant vWF polypeptide comprises a first amino acid sequence present in a reference vWF polypeptide and a second amino acid sequence heterologous to the first, wherein the recombinant vWF polypeptide is capable of binding a FVIII. In one embodiment, an isolated nucleic acid molecule encoding the recombinant vWF polypeptide comprises the nucleotide sequence set forth in SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO : 46.

The polynucleotides of the invention can include variants which have substitutions, deletions, and/or additions which can involve one or more nucleotides. The variants can be altered in coding regions, non-coding regions, or both. Alterations in the coding regions can produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and FVIII binding ability of the polypeptides of the present invention. Further embodiments of the invention include nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence encoding a polypeptide having the amino acid sequences set for therein; and (b) a nucleotide sequence complementary to any of the nucleotide sequences in (a) above.

By a polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence encoding a polypeptide is intended that the nucleotide sequence of the polynucleotide be identical to the reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the polypeptide. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. Two or more polynucleotide sequences can be compared by determining their percent identity. The percent identity of two sequences, whether nucleic acid or peptide sequences, is generally described as the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be extended to use with peptide sequences using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. 0. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6) : 6745-6763 (1986) . An implementation of this algorithm for nucleic acid and peptide sequences is provided by the Genetics Computer Group (Madison, Wis.) in their BESTFIT utility application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, Wis.) .

For example, due to the degeneracy of the genetic code, one of ordinary skill in the art will recognize that a number of the nucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of the nucleic acid sequences described herein can encode the polypeptide . In fact, because degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing any functional assays or measurements described herein. It will be further recognized by the artisan skilled in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having FVIII binding capability. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein binding (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid) .

Recently, advances in the synthetic production of longer polynucleotide sequences have enabled the synthetic production of nucleic acids encoding significantly longer polypeptides without the use of traditional cloning techniques. Commercial providers of such services include

Blue Heron, Inc., Bothell, WA (http://worldwideweb.blueheronbio.com). Technology utilized by Blue Heron, Inc. is described in U.S. Patent Nos .

6, 664, 112; 6, 623, 928; 6, 613, 508; 6, 444, 422; 6,312,393;

4,652,639; U.S. Published Patent Application Nos.

20020119456A1; 20020077471A1 ; and Published International Patent Applications (Publications Nos) WO03054232A3 ;

WO0194366A1; W09727331A2; and WO9905322A1, all incorporated herein by reference.

Of course, traditional techniques of molecular biology, microbiology, and recombinant nucleic acid can also be used to produce the polynucleotides of the invention. These techniques are well known and are explained in, for example, Current Protocols in Molecular Biology, F. M. Ausebel, ed., Vols. I, II and III (1997); Sambrook et al . , Molecular Cloning: A Laboratory Manual, 2^nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989); DNA Cloning: A Practical Approach, D. N. Glover, ed., Vols. I and II (1985); Oligonucleotide Synthesis, M. L. Gait, ed. (1984); Nucleic Acid Hybridization, Hames and Higgins, eds . (1985); Transcription and Translation, Hames and Higgins, eds. (1984); Animal Cell Culture, R. I. Freshney, ed. (1986); Immobilized Cells and Enzymes, IRL Press (1986); Perbal, "A Practical Guide to Molecular Cloning"; the series, Methods in Enzymology, Academic Press, Inc. (1984); Gene Transfer Vectors for Mammalian Cells, J. H. Miller and M. P. Calos, eds., Cold Spring Harbor Laboratory (1987); and Methods in Enzymology, Wu and Grossman and Wu, eds., respectively, Vols. 154 and 155, all incorporated herein by reference .

In other aspects, a nucleotide sequence encoding the recombinant vWF polypeptide of the present is provided, wherein the recombinant vWF polypeptide comprises a first amino acid sequence present in a reference vWF polypeptide and a second amino acid sequence heterologous to the first, wherein the recombinant vWF polypeptide is capable of binding a FVIII. In one embodiment, an isolated nucleic acid molecule encoding the recombinant vWF polypeptide comprises the nucleotide sequence set forth in SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, and SEQ ID NO : 46.

Also provided, in other aspects, are one or more expression vectors comprising the nucleic acid molecules encoding the polypeptides of the present invention. Host cells also are provided that recombinantly express the polypeptides of the present invention. One of skill in the art may make a selection among vectors, expression control sequences and hosts that provide suitable levels of expression of the protein (s) of interest and the a2 , 3-sialyltransferase . For example, in selecting vectors, the host can be considered because the vector must replicate in it or be able to integrate into the chromosome. The vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers, may also be considered. In selecting an expression control sequence, a variety of factors also can be considered. These include, for example, the relative strength of the sequence, its controllability, and its compatibility with the nucleotide sequence encoding the polypeptide. Hosts may be selected by consideration of their compatibility with the chosen vector, the toxicity of the product coded for by the nucleotide sequence, their secretion characteristics, their ability to fold a particular polypeptide correctly, their fermentation or culture requirements, and the ease of purification of the products coded for by the nucleotide sequences. A recombinant vector can be an autonomously replicating vector, i.e. a vector which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid. Alternatively, the vector can be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome ( s ) into which it has been integrated .

In some embodiments, vectors are preferably expression vectors, in which the encoding nucleotide sequence (s) is operably linked to additional segments required for transcription of the nucleotide sequence. A vector is typically derived from plasmid or viral DNA. A number of suitable expression vectors for expression in host cells are commercially available or known to persons of ordinary skill in the art .

Expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus and cytomegalovirus (CMV) . Specific vectors are, e.g., pCDNA3.1 ( + ) Hyg (Invitrogen, Carlsbad, CA) and pCI-neo (Stratagene, La 1, CA) .

Expression vectors for bacterial hosts include, for example, known bacterial plasmids, such as plasmids from E. coli, including pBR322, Novagen® pET3a and pETl2a (Merck KGaA, Darmstadt, Germany), RP4 and phage DNAs, e.g., the numerous derivatives of phage lambda, e.g., NM989, and other DNA phages, such as Ml3 and filamentous single stranded DNA phages .

Expression vectors for yeast cells include, for example, the 2μ plasmid and derivatives thereof, the POTl vector (U.S. Patent No. 4,931,373), the pJS037 vector (described in Okkels, Ann. New York Acad. Sci. 782, 202-207, 1996) and pPICZ A, B or C (Invitrogen, Carlsbad, CA) . Expression vectors for insect cells include, for example, pVL941, pBG311 (Cate et al . , Cell, 45, pp. 685-98 (1986)), pBluebac 4.5 and pMelbac (Invitrogen, Carlsbad, CA) as well as PVL1392 (BD Biosciences (Pharmingen) , San Jose, CA) . Other vectors known in the art include those that allow the encoding nucleotide sequence to be amplified in copy number. They include, for example, vectors able to be amplified by DHFR amplification and glutamine synthetase ("GS") amplification .

The recombinant vector may further comprise a DNA sequence enabling the vector to replicate in the host cell. An example of such a sequence (when the host cell is a mammalian cell) is the SV40 origin of replication. When the host cell is a yeast cell, suitable sequences enabling the vector to replicate are the yeast plasmid 2p replication genes REP 1-3 and origin of replication.

The vector may also comprise a selectable marker, e.g. a gene, the product of which complements a defect in the host cell, such as the gene coding for dihydrofolate reductase (DHFR) or the Schizosaccharomyces pombe TPI gene, or one which confers resistance to a drug, e.g. ampicillin, kanamycin, tetracycline, chloramphenicol, neomycin, hygromycin or methotrexate. For filamentous fungi, selectable markers include amdS, pyrG, arcB, niaD, and sC.

One of ordinary skill in the art knows that the control sequences that are necessary or advantageous for recombinant expression, may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, leader sequence, polyadenylation sequence, propeptide sequence, promoter, enhancer or upstream activating sequence, signal peptide sequence, and transcription terminator.

Examples of suitable control sequences for directing transcription in mammalian cells include the early and late promoters of SV40 and adenovirus, e.g. the adenovirus 2 major late promoter, the MT-1 (metallothionein gene) promoter, the human CMV immediate-early gene promoter, the human elongation factor la (EF-la) promoter, the Drosophila minimal heat shock protein 70 promoter, the Rous Sarcoma Virus (RSV) promoter, the human ubiquitin C (UbC) promoter, the human growth hormone terminator, SV40 or adenovirus Elb region polyadenylation signals and the Kozak consensus sequence .

In other embodiments, a synthetic intron may be inserted in the 5 ' untranslated region of the encoding nucleot ide sequence. An example of a synthetic intron is the synthetic intron from the plasmid pCI-Neo (Promega, Madison, WI) .

Examples of suitable control sequences for directing transcription in insect cells include the polyhedrin promoter, the PlO promoter, the Autographa californica polyhedrosis virus basic protein promoter, the baculovirus immediate early gene 1 promoter and the baculovirus 39K delayed-early gene promoter, and the SV40 polyadenylation sequence.

Examples of suitable control sequences for use in yeast host cells include the promoters of the yeast a-mating system, the yeast triose phosphate isomerase (TPI) promoter, promoters from yeast glycolytic genes or alcohol dehydrogenase genes, the ADH2-4c promoter and the inducible GAL promoter.

Examples of suitable control sequences for use in filamentous fungal host cells include the ADH3 promoter and terminator, a promoter derived from the genes encoding Aspergillus oryzae TAKA amylase, triose phosphate isomerase or alkaline protease, an A. niger a-amylase, A. niger or A. nidulans glucoamylase , A. nidulans acetamidase, Rhizomucor miehei aspartic proteinase or lipase, the TPIl terminator and the ADH3 terminator.

Examples of suitable control sequences for use in bacterial host cells include promoters of the lac system, the trp system, the TAC or TRC system and the major promoter regions of phage lambda.

The nucleotide sequence of the invention, whether prepared by site-directed mutagenesis, synthesis or other methods, may or may not also include a nucleotide sequence that encode a signal peptide. The signal peptide is present when the polypeptide is to be secreted from the cells in which it is expressed. Such signal peptide, if present, should be one recognized by the cell chosen for expression of the polypeptide. The signal peptide may be homologous (e.g. be that normally associated with the expressed product) or heterologous (i.e. originating from another source than the expressed product) to the expressed product or may be homologous or heterologous to the host cell, i.e. be a signal peptide normally expressed from the host cell or one which is not normally expressed from the host cell. Accordingly, the signal peptide may be prokaryotic, e.g. derived from a bacterium such as E. coli, or eukaryotic, e.g. derived from a mammalian, or insect or yeast cell.

The presence or absence of a signal peptide will, e.g., depend on the expression host cell used for the production of the polypeptide, the product to be expressed (whether it is an intracellular or extracellular product) and whether it is desirable to obtain secretion. For use in filamentous fungi, the signal peptide may conveniently be derived from a gene encoding an Aspergillus sp . amylase or glucoamylase , a gene encoding a Rhizomucor miehei lipase or protease or a Humicola lanuginosa lipase. The signal peptide may be derived from a gene encoding A. oryzae TAKA amylase, A. niger neutral a-amylase, A. niger acid-stable amylase, or A. niger glucoamylase . For use in insect cells, the signal peptide may be derived from an insect gene such as the lepidopteran Manduca sexta adipokinetic hormone precursor, the honeybee melittin, ecdysteroid UDPglucosyltransferase (egt), or human pancreatic lipase (hpl) .

In some embodiments, a signal peptide for use in mammalian cells is that of huIFNG or the murine Ig kappa light chain signal peptide. For use in yeast cells, suitable signal peptides include, for example, a-factor signal peptide from S. cerevisiae, the signal peptide of mouse salivary amylase, a modified carboxypeptidase signal peptide, the yeast BARl signal peptide, and the yeast aspartic protease 3 (YAP3) signal peptide.

Examples of signal peptides include, but are not limited to, the sequences as set forth in SEQ ID NOs:50, 51, 52, 53, and 54.

In one embodiment, a propeptide sequence comprises the amino acid sequence as set forth in SEQ ID NO: 55. In another embodiment, the amino acid sequence of a polypeptide comprising a signal peptide sequence contiguous with a propeptide sequence is as set forth in SEQ ID NO: 56, which is encoded by the nucleotide sequence as set forth in SEQ ID NO: 57. Any suitable host may be used to express the a2 , 3-sialyltransferase and the protein (s) of interest (e.g., the FVIII and/or the vWF), including bacteria, fungi (including yeasts), plant, insect, mammal, or other appropriate animal cells or cell lines, as well as transgenic animals or plants. Examples of bacterial host cells include gram positive bacteria such as strains of Bacillus, e.g. B. brevis or B. subtilis, Pseudomonas or Streptomyces, or gram negative bacteria, such as strains of E. coll. The introduction of a vector into a bacterial host cell may, for example, be effected by protoplast transformation, using competent cells, electroporation, or conjugation . Examples of suitable filamentous fungal host cells include strains of Aspergillus, e.g. A. oryzae, A. niger, or A. nidulans, Fusarium or Trichoderma . Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall. Other suitable procedures for transformation of fungal host cells are known in the art.

Examples of suitable yeast host cells include strains of Saccharomyces, e.g. S. cerevisiae, Schizosaccharomyces, Kluyveromyces, Pichia, such as P. pastoris or P. methlanolica, Hansenula, such as H. Polymorpha or Yarrowia. Methods for transforming yeast cells with heterologous DNA and producing heterologous polypeptides therefrom are known in the art including, e.g., as disclosed by Clontech Laboratories, Inc, Palo Alto, Calif., USA (in the product protocol for the Yeastmaker™ Yeast Tranformation System Kit) .

Examples of suitable insect host cells include a Lepidoptora cell line, such as Spodoptera frugiperda (Sf9 or Sf21) or Trichoplusioa ni cells (High Five) .

Examples of suitable mammalian host cells include Chinese hamster ovary (CHO) cell lines, (e.g. CHO-Kl; ATCC CCL-61), Green Monkey cell lines (COS) (e.g. COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651)); mouse cells (e.g. NS/O) , Baby Hamster Kidney (BHK) cell lines (e.g. ATCC CRL-1632 or ATCC CCL-10), and human cells (e.g. HEK 293 (ATCC CRL-1573), AGEl.HN neuronal cell line (ProBioGen, Berlin, Germany), human amniocytes such as, for example, CAP^® (CEVEC, Koln, Germany), as well as plant cells in tissue culture. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection.

Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection, electroporation, DEAE-dextran mediated transfection, liposome-mediated transfection, and viral vectors. These and other methods are well known in the art, e.g. as described by Ausubel et al. (eds.), 1996, Current Protocols in Molecular Biology, John Wiley & Sons, New York, USA.

In the expression/production methods of the present invention, cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, cells may be cultivated by shake flask cultivation, small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known n the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection) . If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates .

The resulting polypeptide may be recovered by methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation.

In some embodiments, wherein insoluble polypeptides are isolated from a host cell (e.g. a prokaryotic host cell), the host cell can be exposed to a buffer of suitable ionic strength to solubilize most host proteins, but in which aggregated polypeptides of interest may be substantially insoluble, and disrupting the cells so as to release the inclusion bodies and make them available for recovery by, for example, centrifugation . This technique is known to one of ordinary skill in the art, and a variation is described, for example, in U.S. Pat. No. 4, 511, 503, which is incorporated by reference herein for its teaching of a method of solubilizing heterologous protein, produced in an insoluble refractile form in a recombinant host cell culture. Without being held to a particular theory, it is believed that expression of a recombinant protein, in e.g. E. coli, may result in the intracellular deposition of the recombinant protein in insoluble aggregates called inclusion bodies. Deposition of recombinant proteins in inclusion bodies can be advantageous both because the inclusion bodies accumulate highly purified recombinant protein and because protein sequestered in inclusion bodies is protected from the action of bacterial proteases.

Generally, host cells (e.g., E. coli cells) are harvested after an appropriate amount of growth and suspended in a suitable buffer prior to disruption by lysis using techniques such as, for example, mechanical methods (e.g., sonic oscillator) or by chemical or enzymatic methods. Examples of chemical or enzymatic methods of cell disruption include spheroplasting, which comprises the use of lysozyme to lyse bacterial wall, and osmotic shock, which involves treatment of viable cells with a solution of high tonicity and with a cold-water wash of low tonicity to release the polypeptides .

Following host cell disruption, the suspension is typically centrifuged to pellet the inclusion bodies. The resulting pellet contains substantially all of the insoluble polypeptide fraction, but if the cell disruption process is not complete, it may also contain intact cells or broken cell fragments. Completeness of cell disruption can be assayed by resuspending the pellet in a small amount of the same buffer solution and examining the suspension with a phase-contrast microscope. The presence of broken cell fragments or whole cells indicates that additional disruption is necessary to remove the fragments or cells and the associated non-refractile polypeptides. After such further disruption, if required, the suspension can be again centrifuged and the pellet recovered, resuspended, and analyzed. The process can be repeated until visual examination reveals the absence of broken cell fragments in the pelleted material or until further treatment fails to reduce the size of the resulting pellet. Once obtained from the solubilized inclusion bodies or at a later stage of purification, the polypeptide can be suitably refolded in a suitable refolding buffer such as those known in the art . The degree of any unfolding can be determined by chromatography including reversed phase-high performance liquid chromatography (RP-HPLC) .

If the recombinantly expressed proteins of the present invention are not already in soluble form before they are to be refolded, they may be solubilized by incubation in a solubilization buffer comprising chaotropic agent (e.g., urea, guanidine) and reducing agent (e.g., glutathione, dithiothreitol (DTT) , cysteine) in amounts necessary to substantially solubilize the polypeptides. This incubation takes place under conditions of protein concentration, incubation time, and incubation temperature that will allow solubilization of the protein to occur. Measurement of the degree of solubilization can be carried out by turbidity determination, by analyzing protein fractionation between the supernatant and pellet after centrifugation on reduced SDS gels, by protein assay (e.g., the Bio-Rad protein assay kit), or by high performance liquid chromatography (HPLC) . The pH of the solubilization buffer can be alkaline, preferably at least about pH 7.5, with the preferred range being about pH 7.5 to about pH 11. The concentration of the protein of the present invention in the buffered solution for solubilization must be such that the protein will be substantially solubilized and partially or fully reduced and denatured. Alternatively, the recombinant protein may be initially insoluble. The exact amount to employ will depend, e.g., on the concentrations and types of other ingredients in the buffered solution, particularly the type and amount of reducing agent, the type and amount of chaotropic agent, and the pH of the buffer. For example, the concentration of recombinant protein can be increased if the concentration of reducing agent, e.g., glutathione, is concurrently increased .

As the skilled artisan will recognize, procedures for purifying recombinant proteins will vary according to such factors as the type of host cells employed and whether or not the proteins are secreted into the culture medium. For example, when expression systems that secrete the recombinant protein are employed, the culture medium first may be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration medium. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, (e.g., silica gel having pendant methyl or other aliphatic groups) can be employed to further purify the recombinantly expressed polypeptide. Some or all of the foregoing purification steps, in various combinations, can be employed to provide a substantially homogeneous recombinant protein.

The proteins of the present invention may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion) , electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J. C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989) .

In some embodiments, suitable moieties may be incorporated into the expression system to facilitate purification of the expressed sialylated protein. A number of heterologous polypeptides are known in the art that can facilitate identification and/or purification of recombinant fusion proteins of which they are a part . Examples include polyarginine , polyhist idine , or HAT™ (Clontech) , which is a naturally-occurring sequence of non-adjacent histidine residues that possess a high affinity for immobilized metal ions. Proteins comprising these heterologous polypeptides can be purified by, for example, affinity chromatography using immobilized nickel or TALON™ resin (Clontech) , which comprises immobilized cobalt ions. Heterologous polypeptides comprising polyarginine allow effective purification by ion exchange chromatography. Other useful heterologous polypeptides include the FLAG™ peptide, which is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant fusion protein. A murine hybridoma designated 4E11 produces a monoclonal antibody that binds the FLAG™ peptide in the presence of certain divalent metal cations. The 4E11 hybridoma cell line has been deposited with the American Type Culture Collection under accession no. HB 9259. Monoclonal antibodies that bind the FLAG™ peptide can be used as affinity reagents to recover a polypeptide purification reagent that comprises the FLAG™ peptide. Other suitable protein tags and affinity reagents include, without limitation, GST-Bind™ system (Novagen) ; T7-Tag™ affinity purification kit (Novagen); or 3) those described in the Strep-tag™ system (Novagen) . Further, fusions of two or more of the tags described above, such as, for example, a fusion of a FLAG tag and a polyhistidine tag, can be also be employed .

In one embodiment, the cells are ElA-expressing cells. Methods for recombinant protein production in ElA-expressing cells are disclosed by, e.g., WO 2000/63403, which is herein incorporated by reference for its teaching of ElA-expressing cells and recombinant expression therein. In some embodiments, the cells are PER.C6® cells (Crucell, Leiden, The Netherlands) .

In some embodiments, the recombinant a2 , 3-sialyltransferase is expressed at the cell surface. A number of methods are known in the art for displaying proteins on the surfaces of cells. For example, pDisplay™ is a commercially available vector that is used to display a polypeptide on the surface of a mammalian cell (Invitrogen, Carlsbad, CA) . In this vector, a multiple cloning site resides between sequences that encode two identifiable peptides, hemagglutinin A and myc epitopes. The vector also includes sequences that encode an N-terminal signal peptide derived from a murine immunoglobulin κ-chain, and a type I transmembrane domain of platelet-derived growth factor receptor, located and the C-terminus . In this way, the expressed product (e.g., the α2 , 3-sialyltransferase) is expressed by a transfected cell as an extracellular fusion protein, anchored to the plasma membrane at the fusion protein C-terminus by the transmembrane domain.

In other embodiments, the recombinant a2 , 3-sialyltransferase and the protein (s) to be sialylated are encoded by respective nucleic acid sequences that are each present in a single or multiple expression vector (s) . For example, in some embodiments, a single expression vector may comprise: (i) a first nucleic acid sequence encoding the recombinant a2 , 3-sialyltransferase; and (ii) a second nucleic acid sequence encoding the recombinant protein. In other embodiments, the polynucleotide encoding the a2 , 3-sialyltransferase is present on a different vector than the vector comprising the polynucleotide ( s ) encoding the recombinant protein (s) to be sialylated.

In still further embodiments, the present invention provides a method for preparing a recombinant FVIII protein, the method comprising: co-expressing the recombinant FVIII protein with a recombinant a2 , 3-sialyltransferase in a mammalian cell under a condition sufficient to increase sialylation of the recombinant protein in the cell.

In some embodiments, the cell co-expresses a recombinant vWF . In one embodiment, the recombinant vWF is expressed as a recombinant vWF polypeptide comprising a propeptide.

In other embodiments, the recombinant vWF is expressed as a recombinant vWF polypeptide comprising a propeptide and an Fc peptide.

In another embodiment, the cell is a PER.C6 cell. In still further aspects, the present invention provides a sialylated protein including compositions comprising said protein. In preferred embodiments, the sialylated protein is prepared in accordance with the methods of the present invention .

In some embodiments, a recombinant FVIII protein of the present invention comprises a higher tyrosine sulfation level or sialyation capping level relative to a FVIII protein expressed by a non-human cell. In one embodiment, the non-human cell is a CHO or BHK cell.

In another embodiment, the recombinant FVIII protein of the present invention comprises a higher tyrosine sulfation level or sialyation capping level relative to Xyntha® (Wyeth Pharmaceuticals Inc., Philadelphia, PA), Kogenate® (Bayer Healthcare Pharmaceuticals Inc., Wayne, NJ) , Advate® (Baxter Healthcare, Deerfield, IL) or all of these. Xyntha®, Kogenate® and Advate® are commercially available antihemophilic factors recombinantly produced in non-human cell lines . In other embodiments, a recombinant FVIII protein of the present invention comprises less than about 10%, illustratively, less than about 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5%, and 0.1% of non-sulfated tyrosine at position 1680 (or equivalent position) to total tyrosine-1680 (or equivalent position) . In some embodiments, a recombinant FVIII protein of the present invention comprises undetectable levels of non-sulfated tyrosine at position 1680 (or equivalent position) . In one embodiment, a composition comprising the recombinant FVIII protein is characterized by 100% Tyr-1680 sulfation of the FVIII proteins present in the composition.

In one embodiment, a recombinant FVIII protein of the present invention has a binding to a vWF protein that exceeds, or is similar to, the high-affinity binding constant (K_d) of plasma-derived FVIII to human vWF (i.e. K_d less than 0.2-0.4 nanomolar), and exceeds the K_d of binding of similar compositions derived from non-human mammalian cells or human cell derivatives not related to PER.C6,

In some embodiments, a recombinant FVIII protein of the present invention comprises a Neu5Gc level that is less than the level of Neu5Gc present on FVIII recombinantly produced in non-human mammalian cells, or in human cells other than PER.C6. In other embodiments, a recombinant FVIII protein of the present invention has undetectable levels of Neu5Gc.

In one embodiment, a recombinant FVIII protein of the present invention lacks Neu5Gc. In another embodiment, a recombinant FVIII protein of the present invention is characterized as a recombinant FVIII that is negative for Neu5Gc and does not elicit an immune response of antibodies specific for Neu5Gc.

In another embodiment, the level of sialic acid present on recombinant proteins of the present invention is at least 70%, 80%, 90%, 95% or greater, sialylation.

In other embodiments, the average FVIII productivity (Qp) of a clonal cell line expressing FVIII in accordance with the methods of the present invention is at least about 5 U/cell/ day, illustratively, at about 5 to about 20, about 6 to about 18, about 8 to about 16, about 10 to about 14 U/cell/ day. In one embodiment, the average FVIII productivity (Qp) is greater than about 2.5 U/cell/ day.

In still further aspects, the present invention provides compositions and methods for treating a subject with the sialylated recombinant proteins of the present invention. The sialylated protein (s) of the invention is/are preferably administered in a composition including a pharmaceutically acceptable carrier or excipient . Generally, the carrier or excipient is suitable for use in patients to whom it is administered. Such pharmaceutically acceptable carriers and excipients are well known in the art (e.g. as disclosed by Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro, Ed., Mack Publishing Company (1990); Pharmaceutical Formulation Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard, Eds., Taylor & Francis (2000); and Handbook of Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000)). The sialylated protein (s) of the present invention can be used "as is" and/or in a salt form thereof. Suitable salts include, but are not limited to, salts with alkali metals or alkaline earth metals, such as sodium, potassium, calcium and magnesium, as well as e.g. zinc salts. These salts or complexes may by present as a crystalline and/or amorphous structure .

The sialylated protein (s) of the invention can be administered at a dose approximately paralleling that employed in therapy with known commercial preparations. The exact dose to be administered depends on the circumstances . Normally, the dose should be capable of preventing or lessening the severity or spread of the condition or indication being treated. It will be apparent to those of skill in the art that an effective amount of the sialylated protein or composition of the invention can depend on a number of factors including, for example, the condition or disease being treated, the dose, the administration schedule, whether the sialylated protein or composition is administered alone or in combination with other therapeutic agents, the serum half-life/functional in vivo half-life of the sialylated protein, and the general health of the patient . The present invention also relates to the sialylated protein (s) according to the present invention or a pharmaceutical composition comprising the sialylated protein (s) according to the present invention for use as a medicament. The invention also relates to the use of the sialylated protein (s) according to the present invention, or a pharmaceutical composition of the invention, for the manufacture of a medicament, a pharmaceutical composition or a kit for the treatment of a condition or disease. Examples of a condition or disease include, without limitation, chronic emphysema, cystic fibrosis, chronic obstructive pulmonary disease, and hemophilia A.

The pharmaceutical composition may be formulated in a variety of forms, including liquid, gel, lyophilized, powder, compressed solid, or any other suitable form. The preferred form will depend upon the particular indication being treated and will be apparent to one of skill in the art. In some embodiments, the sialylated proteins of the invention are preferably formulated as a liquid pharmaceutical composition.

The pharmaceutical composition may be administered orally, subcutaneously, intravenously, intracerebrally, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, intraocularly, or in any other acceptable manner, e.g. using PowderJect or ProLease technology. The formulations can be administered continuously by infusion, although bolus injection is acceptable, using techniques well known in the art, such as pumps or implantation. In some instances the formulations may be directly applied as a solution or spray. The preferred mode of administration will depend upon the particular indication being treated and will be apparent to one of skill in the art.

The pharmaceutical composition of the invention may be administered in combination with other therapeutic agents. These agents may be incorporated as part of the same pharmaceutical composition or may be administered separately from the sialylated protein of the invention, either concurrently or in accordance with any other acceptable treatment schedule. In addition, the sialylated protein or pharmaceutical composition of the invention may be used as an adjunct to other therapies.

By way of example, a pharmaceutical composition is a solution designed for parenteral administration. Although formulations can be provided in liquid form, parenteral formulations may also be provided in frozen or in lyophilized form. In the former case, the composition must be thawed prior to use. The latter form is often used to enhance the stability of the active compound contained in the composition under a wider variety of storage conditions, as it is recognized by those skilled in the art that lyophilized preparations are generally more stable than their liquid counterparts. Such lyophilized preparations are reconstituted prior to use by the addition of one or more suitable pharmaceutically acceptable diluents such as sterile water for injection or sterile physiological saline solution .

In case of parenteral therapeutics, they may be prepared for storage as lyophilized formulations or aqueous solutions by mixing, as appropriate, the sialylated protein having the desired degree of purity with one or more pharmaceutically acceptable carriers, excipients or stabilizers typically employed in the art, for example buffering agents, stabilizing agents, preservatives, isotonic agents, non-ionic detergents, antioxidants and/or other miscellaneous additives.

Buffering agents help to maintain the pH in the range which approximates physiological conditions. Suitable buffering agents for use with the present invention include both organic and inorganic acids and salts thereof such as citrate buffers (e.g., monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-monosodium citrate mixture, etc.), succinate buffers (e.g., succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium fumarate-disodium fumarate mixture, etc.), gluconate buffers (e.g., gluconic acid-sodium glyconate mixture, gluconic acid-sodium hydroxide mixture, gluconic acid-potassium glyuconate mixture, etc. ) , oxalate buffer (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) and acetate buffers (e.g., acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.) . Phosphate buffers, histidine buffers, and trimethylamine salts such as Tris may also be employed.

Preservatives may be added to prevent or reduce microbial growth, and can typically be added in amounts of about 0.2% to about 1% (w/v) , for example. Suitable preservatives for use include, without limitation, phenol, benzyl alcohol, meta-cresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g. benzalkonium chloride, bromide or iodide), hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol .

Isotonic agents may be included to ensure isotonicity of liquid compositions, and include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol . Polyhydric alcohols can be present in an amount of about 0.1% to about 25% by weight, illustratively, about 1% to about 5%, taking into account the relative amounts of the other ingredients. Stabilizers refer to a broad category of excipients which can range in function from a bulking agent to an additive which solubilizes the therapeutic agent or helps to prevent denaturation or adherence to the container wall. Typical stabilizers can be polyhydric sugar alcohols; amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine , glutamic acid, threonine, etc., organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodium thioglycolate , thioglycerol , a-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides; proteins such as human serum albumin, bovine serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose, and polysaccharides such as dextran. Stabilizers can be present in the range of, e.g., about 0.1 to about 10,000 parts by weight based on the active sialylated protein weight.

Wetting agents such as, for example, non-ionic surfactants or detergents may be present to help solubilize the therapeutic agent as well as to protect the therapeutic sialylated protein against agitation-induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the sialylated protein. Suitable non-ionic surfactants include polysorbates (e.g., 20, 80), polyoxamers (e.g., 184, 188), Pluronic™ polyols, polyoxyethylene sorbitan monoethers (e.g., Tween™-20, Tween™-80) .

Additional miscellaneous excipients include bulking agents or fillers (e.g. starch), chelating agents (e.g. EDTA) , antioxidants (e.g., ascorbic acid, methionine, vitamin E) and co-solvents.

The active ingredient ( s ) may also be entrapped in microcapsules prepared, for example, by coascervat ion techniques or by interfacial polymerization, for example hydroxymethylcellulose , gelatin or poly- (methylmethacrylate ) microcapsules, in colloidal drug delivery systems (for example liposomes, albumin microspheres, microemulsions , nano particles and nanocapsules ) or in macroemulsions . Such techniques are disclosed in Remington's Pharmaceutical Sciences, supra.

Parenteral formulations to be used for in vivo administration can be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes .

Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the variant, the matrices having a suitable form such as a film or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly ( 2 -hydroxyethyl-methacrylate ) or poly (vinylalcohol ) ) , polylact ides , copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the ProLease™ technology or Lupron ™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D- (-) -3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for long periods such as up to or over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated polypeptides remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity . Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

For oral administration, the pharmaceutical composition may be in solid or liquid form, e.g. in the form of a capsule, tablet, suspension, emulsion or solution. The pharmaceutical composition is preferably made in the form of a dosage unit containing a given amount of the active ingredient. A suitable daily dose for a human or other mammal may vary widely depending on the condition of the patient and other factors, but can be determined by persons skilled in the art using routine methods.

Solid dosage forms for oral administration may include capsules, tablets, suppositories, powders and granules. In such solid dosage forms, the active compound may be admixed with at least one inert diluent such as sucrose, lactose, or starch. Such dosage forms may also comprise, as is normal practice, additional substances, e.g. lubricating agents such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.

The sialylated recombinant protein (s), in one embodiment, may be admixed with other reagents such as, for example, lactose, sucrose, starch powder, cellulose esters of alkanoic acids, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric and sulphuric acids, acacia, gelatin, sodium alginate, polyvinyl-pyrrolidine, and/or polyvinyl alcohol, and tableted or encapsulated for conventional administration. Alternatively, they may be dissolved in saline, water, polyethylene glycol, propylene glycol, ethanol, oils (such as corn oil, peanut oil, cottonseed oil or sesame oil) , tragacanth gum, and/or various buffers. Other ingredients and modes of administration are well known in the pharmaceutical art. A carrier or diluent may include time delay material, such as glyceryl monostearate or glyceryl distearate alone or with a wax, or other materials well known in the art .

In some embodiments, the pharmaceutical compositions may be subjected to conventional pharmaceutical operations such as sterilization and/or may contain conventional reagents such as, for example, preservatives, stabilizers, wetting agents, emulsifiers, buffers, fillers, etc., e.g. as disclosed elsewhere herein.

Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art, such as water. Such compositions may also comprise reagents such as wetting agents, sweeteners, flavoring agents and perfuming agents. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin (e.g., liniments, lotions, ointments, creams, or pastes) and drops suitable for administration to the eye, ear, or nose.

Formulations suitable for use with a nebulizer, either jet or ultrasonic, can include the protein dissolved in water at a concentration of, e.g., about 0.01 to about 25 mg of the protein per mL of solution, preferably about 0.1 to about 10 mg/mL. The formulation can also include a buffer and a simple sugar (e.g., for protein stabilization and regulation of osmotic pressure) , and/or human serum albumin ranging in concentration from about 0.1 to about 10 mg/ml. Examples of buffers that may be used are sodium acetate, citrate and glycine. Preferably, the buffer will have a composition and molarity suitable to adjust the solution to a pH in the range of about 3 to about 9. Generally, buffer molarities of about 1 mM to about 50 mM can be suitable. Examples of sugars which can be utilized are lactose, maltose, mannitol, sorbitol, trehalose, and xylose, usually in amounts ranging from about 1% to about 10% by weight of the formulation.

The nebulizer formulation may also contain a surfactant to reduce or prevent surface induced aggregation of the sialylated protein caused by atomization of the solution in forming the aerosol. Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitan fatty acid esters. Amounts will generally range from about 0.001% to about 4% by weight of the formulation.

Formulations for use with a metered dose inhaler device will generally comprise a finely divided powder. This powder may be produced by lyophilizing and then milling a liquid variant formulation and may also contain a stabilizer such as human serum albumin. Additionally, one or more sugars or sugar alcohols may be added to the preparation if necessary. Examples include lactose maltose, mannitol, sorbitol, sorbitose, trehalose, xylitol, and xylose. The amount added to the formulation can range from about 0.01 to about 200% (w/w) , preferably from approximately about 1 to about 50%, of the sialylated protein present. Such formulations may then be lyophilized and milled to the desired particle size. The properly sized particles can then be suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane , dichlorodifluoromethane , dichlorotetrafluoroethanol , and 1 , 1 , 1 , 2-tetrafluoroethane , or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant. This mixture is then loaded into the delivery device.

Mechanical devices designed for pulmonary delivery of therapeutic products, include but are not limited to, nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those of skill in the art. Examples of commercially available devices suitable for the practice of this invention are Ultravent nebulizer (Mallinckrodt , Inc., St. Louis, MO); Acorn II nebulizer (Marquest Medical Products, Englewood, CO); Ventolin metered dose inhaler (Glaxo Inc., Research Triangle Park, NC) ; Spinhaler powder inhaler (Fisons Corp., Bedford, MA); the "standing cloud" device (Inhale Therapeutic Systems, Inc., San Carlos, CA) ; AIR inhaler (Alkermes, Cambridge, MA); and AERx pulmonary drug delivery system (Aradigm Corporation, Hayward, CA) . The average current dosage for the treatment of a hemophiliac varies with the severity of the bleeding episode. For example, the average doses administered intravenously may be in the range of: about 40 units per kilogram of FVIII for pre-operative indications, about 15 to about 20 units per kilogram for minor hemorrhaging, and about 20 to about 40 units per kilogram administered over an about 8-hour period for a maintenance dose. Other dosages and regimens can be readily determined by one of ordinary skill in the art of treating hemophiliacs.

In still further aspects, the present invention provides a kit. In some embodiments, the kit comprises one or more reagents for preparing a sialylated protein (s) in accordance with the present invention. For example, in one embodiment, the kit comprises an expression vector useful for recombinant expression of the recombinant protein to be sialylated by an a2 , 3-sialyltransferase . In another embodiment, the expression vector comprises a cloning site for incorporating a nucleic acid sequence of a protein of interest, the vector further comprising a nucleic acid sequence encoding an a2 , 3-sialyltransferase . The kit may further comprise instructions. In other embodiments, the kit comprises separate compartments.

In one embodiment, the kit comprises an expression vector, wherein the expression vector comprises a multiple cloning site for cloning a nucleic acid sequence encoding a protein, wherein the vector further comprises a polynucleotide sequence that encodes an a2 , 3-sialyltransferase , wherein the vector is capable of co-expressing the protein and the a2 , 3-sialyltransferase in a cell.

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

Co-Expression In PER.C6 Mammalian Cells

Cells of clonal cell line 078 (BDD-078 cells) , which are PER.C6 cells expressing a B-domain-deleted FVIII (BDD-FVIII) (SEQ ID NO: 6), were maintained in continuous culture by routine (4 day) passaging in PER-MAb media (Hyclone, Logan, UT) in the presence of neomycin/G418 at 125 g/milliliter . Forty-eight hours prior to electroporation, cells were refreshed by seeding cells at about 1 x 10^s cells/ml in fresh PER-MAb medium without antibiotic selection. On the day of the electroporation, about 5 x 10^s cells were resuspended into 100 microliters of Amaxa Nucleofector® Kit V Solution (Lonza Walkersville Inc., Walkersville, MD) .

For each electroporation, the cell suspension was mixed with about 2 to about 5 micrograms of expression plasmid DNA for expression of: (i) a human alpha-2,3 sialyltransferase having the amino acid sequence as set forth in SEQ ID NO : 2 ; and (ii) a truncated vWF polypeptide as described in Table 1 (See also, e.g., schematic in Figure 1A) .

Table 1: Nucleotide and amino acid sequences for truncated vWF-Fc polypeptides.

In another example (Figure IB) , cells of clonal cell line 078 (BDD-078 cells; vide infra) into which full-length vWF gene (as set forth in SEQ ID NO: 8) had been previously transfected were maintained in continuous culture by routine (4 day) passaging in PER-MAb media (Hyclone, Logan, UT) in the presence of neomycin/G418 at 125 g/milliliter (for selection of transfected FVIII plasmid) and zeocin at 100 g/milliliter (for selection of transfected vWF plasmid) . Forty-eight hours prior to electroporat ion, cells were refreshed by seeding cells at about 1 x 10^s cells/ml in fresh PER-MAb medium without antibiotic selection. On the day of the electroporation, about 5 x 10^s cells were resuspended into 100 microliters of Amaxa Nucleofector® Kit V Solution (Lonza Walkersville Inc., Walkersville, MD) .

The nucleotide sequence portion of a molecule encoding a truncated vWF region or full-length vWF was codon-opt imized using algorithms that account for codon usage, secondary structure, inhibitory sequences, and the like. The Fc DNA region of each molecule, which is derived from IgGi, was not subjected to sequence-optimization. Further, each DNA molecule contains a nucleotide sequence that encodes the signal peptide sequence and, additionally, includes a propeptide amino acid sequence.

Restriction sites included in the synthetic genes were cleaved and re-cloned into the corresponding restriction sites in the plasmid expression vector pcDNA3002Neo (Crucell, Netherlands) . Recombinant DNAs were prepared from clones by the SDS-alkaline lysis method according to commonly known protocols, e.g. as described by Sambrook et al., (1989) Molecular Cloning: A Laboratory Manual, CSH; Jones (1995) Gel Electrophoresis: Nucleic Acids Essential Techniques, Wiley. Restriction enzyme-digested DNAs were fractionated on 1% agarose gels to determine size and identity. Verification of plasmid clone sequences was performed by automated DNA sequencing using fluorescent deoxyribonucleotide primers (Applied Biosystems, Carlsbad, CA) . The prepared plasmid expression vector DNAs encoding the various alpha-2,3 sialyltransferse and vWF constructs were each sterilized by a 70% ethanol wash after precipitation, and resuspended in sterile water to a final concentration of about 0.2 to aboutl.O micrograms per microliter .

The cell mixture was then transferred to the electroporation cuvette of an Amaxa Nucleofector® Device (Lonza Walkersville Inc., Walkersville, MD) ; and a pre-set program X-001 was used to electroporate the DNA into cells. After electroporation, the cell suspension was transferred immediately into pre-warmed (37°C) Mab media (SAFC Biosciences, Lenexa, Kansas) in the well of a 6-well plastic microplate. Cells were incubated without shaking in a humidified incubator chamber set at 37 °C, with 5% CO₂ and 95% humidity.

After approximately 48 hours in culture, each electroporation reaction was removed from the 6-well microplate into a 125 culture flask with 20 ml of fresh MAb media (SAFC Bioscience, Lenexa, Kansas) containing appropriate selection antibiotic (s) : Genet icin/G418 or Zeocin were used at final concentrations 125 or 100 micrograms /ml , respectively; in the case where a2 , 3-sialyltransferase gene was selected independently of FVIII and vWF, hygromycin was used at a final concentration of 50 micrograms /ml . Cells were then cultured at 37°C, in 5% CO₂ and 95% humidity without shaking. After 7-10 days, cells were then adapted to growth in PER-MAb media with shaking and routine passaging to prepare a polyclonal pool of transfected cells. Alternatively, selected cells were prepared for limited dilution cloning as described by, e.g., Harlow, E and Lane, D. (1988) . Antibodies: A Laboratory Manual, pp. 116-117 and pp. 222-223. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Cloned cells were checked regularly for growth and eventually were moved into shake flasks for expansion and growth as suspension cells in PER-MAb medium (Hyclone, Logan, UT) . Clarified supernatants from cell cultures expressing BDD-FVIII and truncated vWF-Fc fusion proteins were prepared by triple centrifugation and separation at 2,500 x g for 7 minutes, followed by 2,500 x g for 11 minutes. Supernatants were then filtered through a Sartobran 150 depth filter (0.45 micrometer), followed by a 0.2 micrometer, cellulose acetate filter (#5231307-H4-00) (Sartorius Stedim Stedim Biotech S.A., Aubagne, France) . Filters were pre-wetted using a pump with 100-200 ml of 20 mM Tris, pH 7.0, followed by cell supernatant, and finally flushed with about 25 ml of 20 mM Tris, pH 7.0. The final filtrate was two-fold-diluted cell supernatant and was the material used for column chromatography. Filtered supernatants were applied to a 5 or 10 milliter Protein A-HiTrap column (part 17-1403-01) (GE Healthcare, Piscataway, NJ) on an AKTA Explorer Chromatography System (GE Healthcare, Piscataway, NJ) . System tubing was pre-flushed with 20mM Tris, pH 7.0, and the Protein A column was washed with five column volumes of 20mM Tris, pH 7.0 to ensure a stable baseline before application of sample to the column. Filtered samples were run through the column at 5 milliliter/min, and the eluate was collected as "flow-through". Once all material was loaded, the Protein A-HiTrap column was additionally washed with five column volumes of 20mM Tris, pH 7.0 until a stable baseline was achieved. At this point, the column was washed with four column volumes of 20mM Tris, pH 7.0 containing 0.1 M CaCl₂ and eluted material was collected.

The FVIII bound to the truncated vWF-Fc fusions was then eluted with 20mM Tris, pH 7.0 containing 0.3 M CaCl₂ until the fraction eluted (approximately three column volumes) . The column was then washed with five additional column volumes of 20mM Tris, pH 7.0. The truncated vWF-Fc proteins remaining bound to protein A were stripped from the column by addition of 250mM Glycine, 150mM NaCl, pH 3.9. Alternative elution methods have been described, e.g., Arakawa et al . , Prot . Expr . Purif., 63:158-163 (2009).

All collected samples from the Protein A-HiTrap column were saved, tested for FVIII activity using the chromogenic and/or clotting assay (described above) and aliquots were prepared for SDS-PAGE electrophoresis. In some cases, for example where FVIII peak activity was detected, the protein was loaded onto PD10 columns (GE Healthcare 45000148) that were pre-washed with 25 ml of desired FVIII storage buffer. Two and one-half milliliters of eluted BDD-FVIII was then applied to each column and the desalted BDD-FVIII was eluted with 3.5 ml FVIII storage buffer. Proteins were then placed at -80°C for long-term storage; and, in some cases, serum albumin was added to 10 mg/ml.

The BDD-FVIII unit activity for each of 44 clones examined, that expressed BDD-FVIII/truncated vWF-Fc/ST3, was determined to be (in g/ml) : 252.3, 290.7, 260.3, 163.2, 215.2, 306.7, 309.3, 397.3, 276.3, 181.7, 165.7, 322.3,

198.7, 134.3, 195.3, 166.3, 325.3, 316.3, 273.3, 47.1, 95,

284.8, 119.5, 288, 306.7, 285, 240, 178, 106.8, 133.7, 206, 176.7, 217.3, 211, 248, 139.3, 289, 329, 205.3, 251, 208, 193.7, 167.7, and 274 (average = 228.1 g/ml). The BDD-FVIII unit activity for each of 22 clones examined, that expressed BDD-FVIII/ full length vWF/ST3, was determined to be (in g/ml) : 217.3, 151.7, 169.3, 237.3, 101.3, 132, 265, 184.7, 81.7, 127, 159.7, 116, 120.3, 136.3, 125, 175, 124.3, 133.3, 168.3, 94.3, 85.4, 134.9, (average = 146.3 g/ml) .

The results show that the relative productivity of BDD-FVIII in clones grown at laboratory scale for an equivalent number of days, co-expressed with truncated vWF-Fc/ST3 produced on average more than 50% greater BDD-FVIII than clones expressing full length vWF/ST3 (average productivity = 228 versus 146 g/ml, respectively) . The average FVIII productivity (Qp) of five PER.C6 clones expressing FVIII in the presence of truncated vWF (SEQ ID NO: 9) and a2 , 3-sialyltransferase (SEQ ID NO:2) is at least about 9 to about 16 μϋ/cell/day, which is greater than similar compositions expressed by other non-human or human cells at levels of approximately 0.1-2.5 μϋ/cell/day.

Example 2

N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) Analysis To characterize the BDD-FVIII expressed by PER.C6 cells as described in Example 1, the Neu5Ac and Neu5Gc content of the BDD-FVIII was compared to two commercially available recombinant FVIIIs known in the art, namely Xyntha® Antihemophilic Factor (recombinantly produced in non-human cell lines) (Wyeth Pharmaceuticals Inc., Philadelphia, PA) ; and Kogenate® FS, Antihemophilic Factor (recombinantly produced in non-human cell lines) (Bayer Healthcare Pharmaceuticals Inc., Wayne, NJ) .

BDD-FVIII, Xyntha®, and Kogenate® were analyzed for sialic acid content by high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) . Briefly, sialic acids were released from each glycoprotein by 0.1 N HC1 hydrolysis. Sialic acids from the samples were collected by ultra-filtration through a 3,000 NMWCO membrane and eluted from a Dionex CarboPac PA-1 HPAEC-PAD column (Dionex; Sunnyvale, Calif.) with a sodium acetate gradient separating two forms of sialic acids, namely Neu5Ac and Neu5Gc. 2-Keto-3-deoxynonic acid (KDN) was used as an internal standard .

HPAEC separation of Neu5Ac, KDN, and Neu5Gc of (A) standard mix; (B) Xyntha®, (C) Kogenate®, and (D) BDD-FVIII is shown in Figure 2. The over-laid chromatogram regions corresponding to retention time (RT) of Neu5Gc is shown in Figure 3. The results demonstrate that there is no Neu5Gc in the recombinantly expressed BDD-FVIII as compared to Xyntha® and Kogenate®, each of which contains Neu5Gc.

Without being held to any particular theory, it is believed that Neu5Gc may contribute to the immunogenicity of proteins having this foreign sialic acid. Example 3

Sialylation Capping Levels

In order to determine the sialylation capping levels of BDD-FVIII (3L bioreactor) relative to a commercially available recombinant FVIII, the sialylation levels for each of 6 clones expressing BDD-FVIII were examined and compared to that of Xyntha®. Sialic Acid Capping Assay

Purified recombinant FVIII was digested by trypsin overnight. Trypsin activity was then killed by adding 10X excess of 4 -Amidinophenylmethanesulfonyl fluoride (APMSF) and incubating at room temperature for 10 min. The digest was cleaned-up and concentrated by C18 spin column. The eluate from the C18 spin column, which contains FVIII peptides, was then dried down completely by centrifugation under vacuum. Immediately before glycosidase digestion, the peptides were re-suspended in phosphate buffer and evenly split into two tubes. Neuraminidase and galactosidase were added in one tube (to release total Gal) and galactosidase only (to release uncapped Gal) was added in the other. The digestion was performed at 37 °C for 4 h and then quenched by heating at 70 °C for 10 min. The released galactose was analyzed and quantitated by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) and a CarboPac PA10 column with an isocratic flow of 20 mM NaOH. The percentage of sialic acid capping was calculated by the following equation: % Sialic acid = 1 - (uncapped Gal / total Gal) . Sialic Acid Characterization

Purified recombinant FVIII was digested by trypsin overnight. Trypsin activity was then inactivated by adding 10X excess of 4-Amidinophenylmethanesulfonyl fluoride (APMSF) and incubating at room temperature for 10 min. The trypsin fragments were separated and concentrated by a C18 spin column. The eluate from the C18 spin column, which contains FVIII peptides, was then dried down completely by centrifugation under vacuum. Acid-forced de-sialylation was performed by incubating with 0.1 M HC1 at 80 °C for 1 h. After the de-sialylation, the sample was dried down completely by centrifugat ion under vacuum, re-suspended in 0.1 % TFA, applied over a pre-conditioned C18 spin column, and washed by 0.1 % TFA. The flow-through and wash, which contain the released sialic acid, are pooled together and dried down. Sialic acid detection was performed by HPAEC-PAD with a CarboPac PA10 column with a gradient flow of mobile phase (100 mM NaOH with 1 M sodium acetate) . 3-Deoxy-d-glycero-d-galacto-2-nonulosonic acid (KDN) was spiked into all samples and used as an internal control.

Results

As shown in Table 2, the recombinant BDD-FVIIIs expressed by each of the six cell lines have higher sialylation capping levels than Xyntha®. Table 2. Sialylation Capping Levels

Without being held to any particular theory, it is believed that increased sialylation can correlate with improved survival of protein in plasma due to likely reduced uptake by as ialogylcoprotein receptors in the liver - as sialic acid content decreases, there is a greater likelihood of elimination of protein from circulation. Example 4

Tyr-1680 Sulfation Analysis

In order to determine the sulfation characteristics of BDD-FVIII relative to commercially available recombinant FVIIIs, the percentages of non-sulfated Tyr-1680 to total Tyr-1680 levels for each of 5 clones expressing BDD-FVIII were examined and compared to that of Xyntha®. As shown in Table 2, the recombinant BDD-FVIIIs expressed by each of the six cell lines have higher sialylation capping levels than Xyntha®, Kogenate® or Advate®.

Tyrl680 Sulfation Quantitation

Relative quantitation of Tyrl680 sulfation was performed by a linear ion trap mass spectrometer (LTQ XL) coupled to an Agilent 1200 HPLC and equipped with an electrospray ionization source. Tryptic peptides of FVIII were resolved by HPLC with a C18 analytical column and electrosprayed directed into the mass spectrometer. The LTQ is set in Selected Ion Monitoring mode targeting ions with m/z 1040 and 1000, which correspond to the sulfated and non-sulfated tryptic peptides containing Tyrl680 (KEDFDIY*DEDENQSPR) , respectively. Relative quantitation is based on peak areas from the SIM plot with ionization efficiency normalized to a factor pre-determined by using standard synthetic peptides of unknown amounts.

Results

As shown in Table 3 and Figure 4, the recombinant BDD-FVIIIs expressed by each of the 5 cell lines have 100% Tyr-1680 sulfation, but Xyntha® and Kogenate® do not.

Percentages of non-sulfated Tyr-1680 to total

Without being held to any particular theory, it is believed that mutation of Tyr-1680 of FVIII eliminates vWF binding.

Example 5

Binding of recombinant BDD-FVIII to vWF

Recombinantly expressed FVIII binding to vWF was assessed in an ELISA format with anti-FVIII antibody. Best fit /non-linear regression analysis was used to derive half-maximal mean binding values (K_d, in pM) .

Recombinant BDD-FVIII proteins were purified from FVIII-vWF-Fc complexes as described in Example 1.

One hundred (100) μΐ of rabbit anti-human vWF (Sigma) diluted 1/1000 in PBS was added to the wells of a 96-well microplate ( Immunlon-2HB) and incubated at 37°C for 1 hour. The wells were then blocked overnight at 4°C with 200 μΐ of 2% BSA in PBS to eliminate non-specific protein interactions with the plate. The plate was washed 4 times with Wash Buffer (0.05% Tween-20 /PBS ) , and 100 μΐ of 10% normal human plasma diluted in 0.05% Tween-20, 0.25% BSA/PBS (Binding Buffer) was added and allowed to incubate overnight at 4 °C. Commercially-prepared vWF proteins were then bound in excess to the anti-human vWF antibodies bound to the microplate wells .

Differing concentrations of the recombinant BDD-FVIII proteins were then added to the microplate wells. The BDD-FVIII proteins from individual cell clones were diluted to 800 ng/ml in Binding Buffer. Dilutions of these proteins were made by addition of ΙΟΟμΙ of protein solution to ΙΟΟμΙ of Binding Buffer in column 1 of the plate, followed by serial two-fold dilution and mixing of 100 μΐ into adjacents wells of each of the 12 columns of the microplate, for a total of 11 dilutions, ranging from 400 ng/ml (2500 pM) to 0.39 ng/ml (2.4 pM) , with the final column reserved for a buffer control. Duplicate dilutions were analyzed for each protein. Proteins were allowed to bind for 1 hour at room temperature, followed by 4 washes with Wash Buffer. At that point, 100 μΐ/well of a 1/1000 dilution of Horseradish Peroxidase labeled sheep anti-human FVIII polyclonal antibody (Affinity Biologicals) was added and incubated for 1 hour at room temperature. The wells were again washed 4 times with Wash Buffer, then 100 μΐ/well of tetramethylbenzidine substrate was added and the plate was allowed to develop until the A₆so of the highest concentration samples reached ~0.9. At that point, 100 μΐ/well of 1M H₃P0₄ was added to stop the reaction and the absorbances of the individual wells were measured at 450 nm. Absorbance values for each concentration were averaged, and the data were entered into GraphPad Prism where they were analyzed by non-linear regression using a saturation binding curve algorithm (hyperbolic curve fit) .

Data were expressed as the concentration which results in half-maximal signal, expressed as K_d. The results are shown in Figure 5.

Claims

1. A method for preparing a recombinant protein, the method comprising:

co-expressing the recombinant protein with a recombinant a2 , 3-sialyltransferase in a mammalian cell under a condition sufficient to increase sialylation of the recombinant protein in the cell.

2. The method of claim 1, wherein the a2,3- sialyltransferase comprises a sequence as set forth in SEQ ID NO: 2.

3. The method of claim 1, wherein the recombinant protein is a FVIII.

4. The method of claim 3, wherein the FVIII comprises a sequence as set forth in SEQ ID NO:3, 4, 5, or 6.

5. The method of claim 3, wherein the cell co-expresses a recombinant vWF .

6. The method of claim 5, wherein the vWF is expressed as a polypeptide comprising a propeptide.

7. The method of claim 5, wherein the vWF is expressed as a polypeptide comprising a propeptide and an Fc peptide.

8. The method of claim 6, wherein the polypeptide comprises a sequence as set forth in SEQ ID NO : 7 , 8, 9, 10, 21, 22, 23, 24, 25, 26, or 34.

9. The method of claim 1, wherein the recombinant protein is a vWF .

10. The method of claim 9, wherein the vWF comprises a sequence as set forth in SEQ ID NO : 7 , 8, 9, 10, 21, 22, 23, 24, 25, 26, or 34.

11. The method of claim 9, wherein the cell co-expresses a recombinant FVIII.

12. The method of claim 11, wherein the FVIII comprises a sequence as set forth in SEQ ID NO:3, 4, 5, or 6.

13. A recombinant FVIII protein prepared by the method of claim 1.

14. A recombinant vWF protein prepared by the method of claim 1.

15. A pharmaceutical composition comprising the recombinant protein of claim 13.

16. A pharmaceutical composition comprising the recombinant protein of claim 14.

17. A mammalian cell co-expressing the recombinant FVIII protein of claim 13 and an a2 , 3-sialyltransferase .

18. A mammalian cell co-expressing the recombinant vWF protein of claim 14 and an a2 , 3-sialyltransferase .

19. A cell culture comprising the cell of claim 17.

20. A cell culture comprising the cell of claim 18.

21. A method for treating a blood disorder in a mammal, the method comprising administering the composition of claim 15 to the mammal.

22. A method for treating a blood disorder in a mammal, the method comprising administering the composition of claim 16 to the mammal.

23. A recombinant FVIII protein comprising a higher tyrosine sulfation level or sialyation capping level relative to a FVIII protein expressed by a non-human cell.

24. A composition comprising a recombinant FVIII protein, wherein less than about 5% of the FVIII that are present in the composition are non-sulfated at tyrosine 1680 or equivalent position.

25. A composition comprising a recombinant FVIII protein, wherein about 100% of the FVIII that are present in the composition are sulfated at tyrosine 1680 or equivalent position .

26. A recombinant FVIII protein comprising a Neu5Gc level that is less than the level of Neu5Gc present on a FVIII recombinantly produced in a non-human cell.

27. A recombinant FVIII protein lacking Neu5Gc.