HK1168545A - Lyophilized recombinant vwf formulations - Google Patents

Description

Lyophilized recombinant VWF formulations

Technical Field

The present invention relates generally to formulations of lyophilized recombinant VWF and methods of producing lyophilized compositions comprising recombinant VWF. Background

Von Willebrand Factor (VWF) is a glycoprotein that circulates in plasma in a range of multimeric forms ranging in size from about 500 to 20,000 kD. The multimeric form of VWF consists of 250kD polypeptide subunits linked by disulfide bonds. VWF mediates the initial platelet adhesion to the subendothelial membrane of the damaged vessel wall. Only the larger polymers exhibit hemostatic activity. It is speculated that endothelial cells secrete larger polymeric forms of VWF, and those forms of VWF with low molecular weight (low molecular weight VWF) are produced by proteolytic cleavage. Polymers of large molecular mass are stored in the Wobuel-palade bodies of endothelial cells (Weibel-Pallade bodies) and are released upon stimulation.

VWF is synthesized by endothelial cells and megakaryocytes as a prepro-VWF (prepro-VWF), which largely consists of repetitive domains. Once the signal peptide is cleaved, the pro-VWF dimerizes via disulfide linkage at its C-terminal domain. The dimer serves as a protomer for multimerization, which is controlled by a disulfide bond linkage between the free ends. After assembly into multimers, the propeptide sequence is proteolytically removed (Leyte et al, biochem. J.274(1991), 257-261).

The primary translational product predicted from the cDNA of cloned VWF is a precursor polypeptide with 2813 residues (pre-precursor VWF). The prepro-VWF consists of a 22 amino acid signal peptide and a 741 amino acid propeptide, the mature VWF comprising 2050 amino acids (Ruggeri ZA, and Ware, j., faeb j., 308-316 (1993)).

A drawback of VWF is the etiology of Von Willebrand Disease (VWD), which is characterized by a more or less pronounced bleeding phenotype. VWD type 3 is the most severe form, with complete absence of VWF, whereas VWD type 1 involves a quantitative loss of VWF, the phenotype of which can be very mild. VWD type 2 involves a defect in the nature of VWF and can be as severe as VWD type 3. VWD type 2 has many subtypes, some of which are associated with loss or reduction of high molecular weight multimers. Von willebrand disease type 2A (VWS-2A) is characterized by the simultaneous loss of medium and large multimers. VWS-2B is characterized by the loss of the highest molecular weight multimer. Other diseases and disorders associated with VWF are known in the art.

U.S. patent nos. 6,531,577, 7,166,709 and european patent application No.04380188.5 describe formulations of VWF of plasma origin. However, in addition to the problems of quantity and purity with plasma-derived VWF, there is also a risk of blood-borne pathogens such as viruses and variant creutzfeld-jakob disease (vCJD). Furthermore, VWF is known to form aggregates under stress conditions.

Therefore, there is a need in the art to develop a stable pharmaceutical formulation comprising recombinant VWF. Disclosure of Invention

The present invention provides formulations useful for lyophilized recombinant VWF, resulting in a highly stable pharmaceutical composition. For those disorders or conditions that would benefit from administration of recombinant VWF, the stable pharmaceutical composition can be used as a therapeutic agent to treat an individual afflicted with such a disorder or condition.

In one embodiment, provided is a pharmaceutical formulation of stable lyophilized recombinant von willebrand factor (rVWF) comprising: (a) rVWF; (b) one or more buffering agents; (c) more than one amino acid; (d) one or more stabilizers; and (e) one or more surfactants; the rVWF comprises a polypeptide selected from the group consisting of: a) SEQ ID No.: 3; b) a biologically active analog, fragment or variant of said a); c) a polypeptide consisting of SEQ ID No.: 1; d) a biologically active analog, fragment or variant of said c); and e) a nucleic acid sequence that hybridizes under moderately stringent hybridization conditions to SEQ ID No.:1 to a polynucleotide of the polynucleotide set forth in (1); the buffer comprises a pH buffer at a concentration in the range of about 0.1mM to about 500mM and a pH in the range of about 2.0 to about 12.0; the amino acid is at a concentration of about 1 to about 500 mM; the stabilizer is at a concentration of about 0.1 to about 1000 mM; and the surfactant concentration is from about 0.01g/L to about 0.5 g/L.

In another embodiment, the rVWF comprises SEQ ID No.: 3. In yet another embodiment, the buffer is selected from the group consisting of citrate, glycine, histidine, HEPES, Tris, and combinations thereof. In another embodiment, the buffer is citrate. In various embodiments, the pH is in the range of about 6.0 to about 8.0, about 6.5 to about 7.5, or about 7.3. In another embodiment, the pH is about 7.3.

In another embodiment, the amino acid is selected from the group consisting of glycine, histidine, proline, serine, alanine, arginine. In another embodiment, the amino acid is at a concentration of about 0.5mM to about 300 mM. In yet another embodiment, the amino acid is glycine at a concentration of about 15 mM.

In one embodiment of the invention, the rVWF comprises SEQ ID No.: 3; wherein the buffer is citrate and has a pH of about 7.3; and wherein the amino acid is glycine at a concentration of about 15 mM.

In still another embodiment of the present invention, the one or more stabilizers are selected from the group consisting of mannitol, lactose, sorbitol, xylitol, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, and combinations thereof. In one embodiment, the stabilizing agent is trehalose at a concentration of about 10g/LmM and mannitol at a concentration of about 20 g/L.

In still another embodiment of the present invention, the above surfactant is selected from the group consisting of digitonin, TritonX-100, Triton X-114, Tween 20, Tween 80 and combinations of these surfactants. In yet another embodiment, the surfactant is about 0.01g/L of Tween 80.

In another embodiment of the invention, rVWF comprises SEQ ID No.: 3; wherein the buffer is about 15mM citrate and has a pH of about 7.3; wherein the amino acid is glycine at a concentration of about 15 mM; wherein the stabilizer is trehalose at a concentration of about 10g/L and mannitol at a concentration of about 20 g/L; and wherein the surfactant is about 0.1g/L of Tween 80. Drawings

FIG. 1 shows an ANCOVA analysis of the combined VWF: RCo activity of batches for stability assessment (stored at 5 ℃. + -. 3 ℃).

FIG. 2 shows the increase in residual moisture in rVWF FDP stored at 5 ℃. + -. 3 ℃.

FIG. 3 shows the increase in residual moisture in rVWF FDP stored at 40 ℃. + -. 2 ℃. Detailed description of the preferred embodimentsin definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide the skilled artisan with a general definition of many of the terms used in the present invention: singleton, et al, DICTIONARY OF MICROBIOLOGY AND MOLECULARBIOLOGY (DICTIONARY OF MICROBIOLOGY AND molecular biology, second edition, 1994); THE Cambridge diagnostics OF SCIENCE AND TECHNOLOGY (Cambridge scientific dictionary, Walker edition, 1988); THE GLOSSARY OF GENETICS (GLOSSARY OF GENETICS, fifth edition, R.Rieger et al, Springer Verlag, 1991); and THE HARPER COLLINS DICTIONARY OFBIOLOGY by Hale and Marham (biologies 1991).

Each of the publications, patent applications, patents, and other references cited herein is incorporated by reference in its entirety and for all purposes to the extent that they are not inconsistent with this disclosure.

It should be noted herein that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

The following terms as used herein are intended to have their given meanings unless otherwise specified.

With respect to peptidic compounds, the term "comprising" means that a compound may include additional amino acids at one or both of the amino terminus and the carboxy terminus of a given sequence. Of course, these additional amino acids should not significantly interfere with the activity of the compound. For the compositions of the present invention, the term "comprising" means that the composition may include other components. These components should not significantly interfere with the activity of the composition.

The term "pharmacologically active" means that a substance described herein is determined to have activity that affects a medical parameter (e.g., without limitation, blood pressure, blood cell count, cholesterol level) or a disease state (e.g., without limitation, cancer, autoimmune disease).

The term "expression" as used herein refers to the production of a protein by causing or causing the visualization of information in a gene or DNA sequence, for example, by activating cellular functions associated with the transcription and translation of the corresponding gene or DNA sequence. The expressed DNA sequence is expressed in or by a cell to form an "expression product," such as a protein. The expression product itself, such as the protein formed, may also be said to be "expressed". Expression products can be distinguished as intracellular, extracellular or secreted. The term "intracellular" refers to within a cell. The term "extracellular" refers to outside a cell, such as a transmembrane protein. A substance is "secreted" by a cell if it occurs in significant amounts from some location on or within the cell to the exterior of the cell.

The term "polypeptide" as used herein refers to a polymer composed of amino acid residues, structural variants, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof, joined by peptide bonds. Synthetic polypeptides can be prepared, for example, using an automated polypeptide synthesizer. The term "protein" generally refers to a larger polypeptide. The term "peptide" generally refers to shorter polypeptides.

As used herein, a "fragment" of a polypeptide refers to any portion of any polypeptide or protein that is smaller than the full-length polypeptide or protein expression product.

The term "analog" as used herein refers to any two or more polypeptides, either whole molecules or fragments thereof, that are substantially identical in structure and have the same biological activity but may have different degrees of activity. The amino acid sequence of the analogs differ in composition based on one or more mutations involving substitution, deletion, insertion, and/or addition of one or more amino acids to other amino acids. Substitutions may be conservative or non-conservative based on the degree of physicochemical or functional correlation between the amino acid being substituted and the amino acid with which it is substituted.

As used herein, "variant" means that the polypeptide, protein, or analog thereof is modified to include additional chemical moieties not normally a part of the molecule. These moieties may modulate the solubility, absorption, biological half-life, etc., of the molecule. Alternatively, these moieties may reduce the toxicity of the molecule and eliminate or mitigate any undesirable side effects of the molecule, and the like. Suitable portions for intervention in these effects are disclosed in Remington's Pharmaceutical Sciences (1980). Procedures for coupling these moieties to molecules are known in the art. For example, and without limitation, in one aspect, the variant is a coagulation factor with a chemical modification that confers a longer in vivo half-life to the protein. In other aspects, the polypeptide is modified by glycosylation, pegylation, and/or polysialylation. Recombinant VWF

The polynucleotide and amino acid sequences of the prepro VWF consist of SEQ ID No:1 and SEQ ID No: 2 and are available in GenBank accession nos. NM _000552 and NP _000543, respectively. The amino acid sequence corresponding to the mature VWF protein consists of SEQ ID No:3 (corresponding to amino acids 764-2813 of the full-length prepro-VWF amino acid sequence).

One useful form of rVWF has at least in vivo stabilizing properties, e.g., binds at least one factor viii (fviii) molecule, and optionally has a pharmacologically acceptable glycosylation pattern. Specific examples thereof include VWF which is free of the A2 domain and thus resistant to proteolysis (Lankhof et al, Thromb. Haemost.77: 1008-1013, 1997), and a VWF fragment from Val 449 to Asn 730 which includes the lb domain of glycoprotein and a binding site for collagen and heparin (Pietu et al, biochem. Biophys. Res. Commun.164: 1339-1347, 1989). In one aspect, the determination of the ability of VWF to stabilize at least one FVIII molecule is performed in a VWF deficient mammal according to methods known in the art.

The rVWF of the present invention can be produced by any method known in the art. A specific example is disclosed in WO86/06096 published at 10/23 in 1986 and in us patent application 07/559,509 filed at 7/23 in 1990, the method of producing rVWF referred to therein being incorporated herein by reference. Thus, the methods used are known in the art with the following steps: (i) production of recombinant DNA by genetic engineering, for example by reverse transcription of RNA and/or DNA amplification; (ii) introducing the recombinant DNA into prokaryotic or eukaryotic cells by transfection, e.g., by electroporation or microinjection; (iii) culturing the transfected cells, e.g., in a continuous or batch mode; (iv) (iv) expressing VWF, e.g. constitutively or inducibly, and (v) isolating VWF, e.g. from the culture medium or by collecting the transfected cells, such that (vi) purified rVWF is obtained, e.g. by anion exchange chromatography or affinity chromatography. In one aspect, the recombinant VWF is produced in a transformed host cell using recombinant DNA techniques well known in the art. For example, a sequence encoding a polypeptide may be excised from the DNA with an appropriate restriction enzyme. Alternatively, in another aspect, the DNA molecule may be synthesized using chemical synthesis techniques, such as the phosphoramidite method. Further, in yet another aspect, these techniques are used in combination.

The invention also provides vectors encoding the polypeptides of the invention in suitable hosts. The vector comprises a polynucleotide encoding the polypeptide operably linked to suitable expression control sequences. Methods for effecting such operative ligation prior to or after insertion of the polynucleotide into a vector are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosome binding proteins, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved in transcriptional or translational control. The resulting vector with the polynucleotide is used to transfect a suitable host. Such transfection may be performed by methods known in the art.

Any of a number of well-known host cells are available for use in the practice of the present invention. The choice of a particular host depends on several factors that are recognized in the art, including, for example, compatibility with the chosen expression vector, toxicity of the peptide encoded by the DNA molecule, transfection efficiency, ease of recovery of the peptide, expression characteristics, biosafety, and cost. In achieving a balance of these factors it must also be understood that not all host cells are equally effective for expressing a particular DNA sequence. Among these general guidelines, useful microbial hosts include, but are not limited to: cultured bacterial, yeast and other fungal, insect, plant, mammalian (including human) cells, or other hosts known in the art.

The transfected host cells are cultured under conventional fermentation conditions to allow expression of the desired compound. These fermentation conditions are known in the art. Finally, the polypeptide is purified from the culture medium or the host cell itself by methods well known in the art.

Carbohydrate (oligosaccharide) groups may optionally be attached to known glycosylation sites on proteins, depending on the host cell used to express the compounds of the invention. In general, in the Asn-X-Ser/Thr sequence, the O-linked oligosaccharide is attached to a serine (Ser) or threonine (Thr) residue, while the N-linked oligosaccharide is attached to an asparagine (Asn) residue, where X can be any amino acid other than proline. X is preferably one of the 19 natural amino acids other than proline. The structure of N-linked oligosaccharides and O-linked oligosaccharides and the sugar residues found in each type are different. One carbohydrate type found in both N-linked and O-linked oligosaccharides is N-acetylneuraminic acid (known as sialic acid). Sialic acids are typically the terminal residues of both N-linked and O-linked oligosaccharides, and in one aspect sialic acid imparts acidic properties to the glycosylated compound by virtue of its own negative charge. Such sites may be introduced into the linker of the compounds of the invention and are preferably glycosylated by cells (e.g. in mammalian cells such as CHO, BHK, COS) in the recombinant production of the polypeptide compound. In other aspects, these sites are glycosylated by synthetic or semi-synthetic processes known in the art.

Alternatively, the compounds may be prepared by synthetic means using, for example, solid phase synthesis techniques. Suitable techniques are well known in the art and include Merrifield, published in 1973 at pages 335-61 of chem.polypetides (Katsoyannis and Panayotis eds.), Merrifield, published in 1963 at J.am.chem.Soc.85: 2149. davis et al, biochem intl.10, 1985: 394-414, Stewart and Young was published in Solid Phase Peptide Synthesis in 1969, U.S. Pat. No. 3,941,763, Finn et al was published in The Proteins (third edition) 2 in 1976: 105-: 257, 527. Solid phase synthesis is the preferred technique for producing a single peptide because it is most cost effective in a process for producing small molecule peptides. VWF fragments, variants and analogs

Methods for making polypeptide fragments, variants, or analogs are well known in the art.

Fragments of a polypeptide are those that are produced by, but are not limited to, enzymatic cleavage (e.g., trypsin, chymotrypsin) or by recombinant means to produce a polypeptide having a particular amino acid sequence. The polypeptide fragment produced may comprise a domain of the protein having a specific activity, such as a multimerization domain or any other identifiable VWF domain known in the art.

Methods for preparing polypeptide analogs are also well known. Amino acid sequence analogs of a polypeptide can be substituted, inserted, added, or deleted analogs. Deletion analogs include fragments of the polypeptide lacking one or more residues that are not essential to functional or immunological activity possessed by the native protein. Insertion-type analogs involve the addition of, for example, amino acids at non-terminal sites of the polypeptide. Such analogs may include, for example, but are not limited to, insertion of an immunoreactive epitope or just a residue. Additive analogs include fragments of the polypeptide, including the addition of one or more amino acids at either or both ends of the protein, and include, for example, fusion proteins. Combinations of the above analogs are also contemplated.

Substituted analogs typically replace wild-type amino acids with other amino acids at one or more positions in a protein, and may also be designed to modulate one or more properties of a polypeptide without complete loss of other function or property. In one aspect, the substituted analog is a conservative substitution. A "conservative amino acid substitution" is a substitution of an amino acid with a side chain or amino acid of similar chemical nature. Similar amino acids that undergo conservative substitutions include those with acidic side chains (glutamic acid, aspartic acid), basic side chains (arginine, lysine, histidine), polar amide side chains (glutamine, asparagine), hydrophobic aliphatic side chains (leucine, isoleucine, valine, alanine, glycine), aromatic side chains (phenylalanine, tryptophan, tyrosine), small side chains (glycine, alanine, serine, threonine, methionine), or aliphatic hydroxyl side chains (serine, threonine).

In one aspect, the analogue is substantially homologous or substantially identical to the recombinant VWF on which it is derived. Analogs include those that retain at least some of the biological activity of the wild-type polypeptide, e.g., clotting activity.

Contemplated polypeptide variants include, but are not limited to, polypeptides chemically modified by techniques such as ubiquitination, glycosylation including polysialylation, conjugation to therapeutic or diagnostic agents, labeling, covalent attachment of polymers such as pegylation (polyethylene glycol derivatization), introduction of non-hydrolytic bonds, and insertion or substitution of chemically synthesized amino acids not normally found in human proteins such as ornithine. Variants retain the same or substantially the same binding characteristics as the unmodified molecules of the invention. Such chemical modification may comprise binding an agent to the VWF polypeptide, either directly or indirectly (e.g., via a linker). If indirectly attached, the linker is contemplated to be hydrolyzable or non-hydrolyzable.

In one aspect, preparation of a pegylated polypeptide analog will comprise: step (a) reacting the polypeptide with a polyethylene glycol (e.g. a reactive ester or an aldehyde derivative of a polyethylene glycol) under conditions such that the polypeptide to be linked is capable of associating with one or more PEG groups, and step (b) obtaining the reaction product. Generally, the optimal reaction conditions for the acylation reaction are determined according to known parameters and the desired result. For example, PEG: the greater the ratio of proteins, the higher the percentage of pegylated product. In some embodiments, the constructs to be combined have a single polyethylene glycol moiety at the N-terminus. Polyethylene glycol (PEG) may be conjugated to coagulation factors, for example, to provide a longer half-life in vivo. The PEG group may be of any convenient molecular weight, linear or branched. The average molecular weight of the PEG is between about 2kDa (daltons) and about 100kDa, between about 5kDa and about 50kDa, or between about 5kDa and about 10 kDa. In certain aspects, attachment of the PEG group to the coagulation factor is achieved by acylation or reductive alkylation of a native or engineered reactive group (e.g., aldehyde, amino, sulfhydryl, or ester group) on the PEG moiety with a reactive group (e.g., aldehyde, amino, or ester group) on the coagulation factor, or by any other technique known in the art.

U.S. patent publication No. 20060160948, Fernandes et Gregoriadis; biochim, biophysis, acta 1341: 26-34, 1997 and Saenko et al, haempoilia 12: 42-51, 2006. Briefly, a solution containing 0.1M NaIO was stirred at room temperature in the dark₄The polysialic acid (CA) solution oxidizes CA. The activated CA solution is dialyzed against e.g. 0.05M sodium phosphate buffer pH 7.2 in the dark and the dialyzed solution is added to the rVWF solution and incubated at room temperature in the dark for 18 hours with gentle shaking. Optionally, the free agent is separated from the rVWF-polysialic acid conjugate, for example by ultrafiltration/diafiltration. Conjugation of rVWF to polysialic acid was achieved using glutaraldehyde as a cross-linker (Migneault et al, Biotechniques 37: 790-796, 2004).

In another embodiment, it is further contemplated that the polypeptide of the invention is a fusion protein and the second agent of fusion is a polypeptide. In one embodiment, the polypeptide second agent is not limited to an enzyme, growth factor, antibody, cytokine, chemokine, cell surface receptor, extracellular domain of a cell surface receptor, cell adhesion molecule, or fragment or active domain of the above proteins. In a related embodiment, the second agent is a coagulation factor, such as factor viii (factorviii), factor vii (factorvii), factor ix (factor ix). Fusion proteins can be contemplated to be made using chemical or recombinant techniques well known in the art.

In another embodiment, it is also contemplated that pre-pro-VWF and pro-VWF polypeptides will provide therapeutic benefits in the formulations of the present invention. For example, U.S. patent No.7,005,502 describes that a pharmaceutical preparation comprising a large amount of pro-VWF can induce thrombin generation in vitro. In addition to recombinant, biologically active fragments, variants, or other analogs of naturally occurring mature VWF, the present invention also contemplates the use of pre-pro VWF (shown in SEQ ID NO: 2) or recombinant biologically active fragments, variants, or analogs of pro-VWF (amino acid residues 23 to 764 of SEQ ID NO: 2) in the formulation.

Polynucleotides encoding fragments, variants, and analogs can be readily generated by one of skill in the art to encode biologically active fragments, variants, or analogs of naturally occurring molecules, the resulting products having the same or similar biological activity as the naturally occurring molecule. In various embodiments, these polynucleotides are made using PCR techniques, digestion/ligation of DNA encoding the molecule, and the like. Thus, one skilled in the art can generate single base changes on a DNA strand using any method known in the art, resulting in codon changes and missense mutations. The phrase "moderately stringent hybridization conditions" as used herein means, for example, hybridization in 50% formamide at 42 ℃ and washing in 0.1 XSSC, 0.1% SDS at 60 ℃. Those skilled in the art will appreciate that variations in these conditions occur depending on the length of the sequence to be hybridized and the GC nucleobase content. The standard of rules in the art are appropriate for determining the exact hybridization conditions. See, for example, Molecular Cloning, Sambrook et al, 9.47-9.51 (Cold spring harbor laboratory Press, Cold spring harbor, N.Y., 1989). Freeze drying

In one aspect, the formulation comprising a VWF polypeptide of the invention is lyophilized prior to administration. Lyophilization was performed using techniques common in the art and should be optimized for the composition developed [ Tang et al, Pharm res.21: 191-200, (2004) and Chang et al, Pharm res.13: 243-9(1996)].

In one aspect, the lyophilization cycle consists of three steps: freezing, primary drying, and secondary drying [ APMackenzie, Phil Trans R Soc London, Ser B, Biol 278: 167(1977)]. In the freezing step, the solution is cooled in order to initiate ice formation. In addition, this step causes crystallization of the filler. Sublimation of ice during the primary drying stage is carried out by using a vacuum and introducing heat to promote sublimation and reduce the pressure in the chamber below the freezing point and vapor pressure. Finally, in a secondary drying stage, the adsorbed or bound water is removed at a reduced pressure in the chamber and at an elevated storage temperature. This process produces what is known as a lyophilized cake. Thereafter, the lyophilized cake can be reconstituted with sterile water or a suitable diluent for injection.

The lyophilization cycle not only determines the final physical state of the excipient, but also affects other parameters such as reconstitution time, appearance, stability, and final moisture content. The structure of the composition in the frozen state, which may be used to understand and optimize the lyophilization process, undergoes several transformations (e.g., glass transition, wetting, and crystallization) that occur at a particular temperature. The glass transition temperature (Tg and/or Tg') may provide information about the physical state of the solute and may be determined by Differential Scanning Calorimetry (DSC). Tg and Tg' are important parameters that must be considered in designing the lyophilization cycle. For example, Tg' is important for primary drying. Furthermore, in the dry state, the glass transition temperature provides information on the storage temperature of the final product. Formulation and general excipients

Excipients are additives that confer or enhance the stability and release properties of a pharmaceutical product (e.g., a protein). Regardless of the reason for its inclusion, excipients become an integral part of the formulation and therefore need to be safe and well tolerated by patients. For protein drugs, the choice of excipients is particularly important because they affect the therapeutic efficacy and immunogenicity of the drug. Therefore, protein formulations need to be developed with the proper choice of excipients to provide suitable stability, safety, marketability.

In one aspect, the lyophilized dosage form consists of at least one or more buffers, bulking agents, and stabilizers. In this regard, the use of surfactants is evaluated and selected when aggregation becomes a problem during the lyophilization step or reconstitution process. Suitable buffers are included to maintain the formulation in a stable pH range during lyophilization. A comparison of excipient components for liquid and lyophilized protein formulations is provided in table a.

Table a: excipient components for lyophilized protein formulations

A major challenge in the development of protein formulations is to stabilize the product against the stresses of manufacture, transport and storage. The role of the dosage form excipients is to provide stabilization against these stresses. Excipients are also used to reduce the viscosity of high concentration protein formulations to facilitate their delivery and to enhance patient convenience. In general, excipients can be classified according to the mechanism by which they stabilize proteins against various chemical and physical stresses. Some excipients are used to mitigate the effects of a particular stress or to modulate a particular susceptibility of a particular protein. Other excipients have a broader impact on the physical and covalent stability of the protein. The excipients described herein are arranged according to their chemical type or their functional role in the formulation. In discussing each excipient type, a brief description of its stable mode is provided.

Based on the teachings and guidance provided herein, one of skill in the art will know what amount or range of excipients may be included in any particular formulation to facilitate retention of stability of the biopharmaceutical (e.g., protein) in order to achieve the biopharmaceutical formulation of the present invention. For example, the amount and type of salt included in the biopharmaceutical formulation of the present invention is selected based on the desired osmolarity of the final solution (i.e., isotonic, hypotonic, or hypertonic) and the amount and osmolarity of other components included in the formulation.

For example, isotonicity can be achieved by adding about 5% sorbitol, whereas about 11% sucrose excipient is required to achieve isotonicity. The selection of the amount or concentration range of the one or more excipients to be included in the biopharmaceutical formulation of the present invention has been exemplified above with salts, polyols and sugars. However, it will be understood by those skilled in the art that the considerations described herein and the specific excipient examples further referenced apply equally to all types and combinations of excipients, including, for example, salts, amino acids, other hypertonicity agents, surfactants, stabilizers, bulking agents, cryoprotectants, lyoprotectants, chelating agents, and/or preservatives.

Furthermore, if reported as molar concentrations at a particular excipient, one skilled in the art will recognize that equivalent percent (%) w/v of solution (e.g., (grams of material in solution sample/ml of solution) × 100%) is also contemplated.

Of course, one of ordinary skill in the art will recognize that the concentrations of excipients described herein are interdependent in a particular formulation. For example, the concentration of bulking agent may be lower when, for example, there is a higher protein concentration or when, for example, there is a higher concentration of stabilizing agent. In addition, one of ordinary skill in the art will recognize that in the absence of a filler, the concentration of the stabilizer may be adjusted accordingly (i.e., using a "tonicity" dosage of the stabilizer) in order to maintain isotonicity of the particular formulation. Conventional Excipients are known in the art and can be obtained from the company of Excipients, firm particulate Formulations (1998), PDA j. pharm. sci. technology, 52: 238-. Buffer and buffer

The stability of pharmacologically active protein formulations is usually observed to be greatest over a narrow pH range. The optimal pH range for this stability needs to be determined in a pre-formulation study. Several approaches such as accelerated stability studies and specific heat screening studies are useful for this attempt (Remmele r.l. jr., et al, Biochemistry, 38 (16): 5241-7 (1999)). Once the formulation is determined, the protein must be produced and maintained over its shelf life. Thus, buffers are almost always used to control the pH of the formulation.

The buffering capacity of the buffer system is greatest at a pH equal to the pKa value and decreases with increasing or decreasing pH. If the pH is within 1 unit of the pKa, there is ninety percent buffering capacity. The buffering capacity also increases proportionally with increasing buffer concentration.

There are several factors to consider when selecting a buffer. The first and most important, the buffer system and its concentration need to be determined according to the pKa and the desired pH of the formulation. It is also important to ensure that the buffer system is compatible with proteins and other dosage form excipients and does not catalyze any degradation reactions. A third important aspect to consider is the perception of irritation and irritation that buffers may cause upon administration. For example, citrate is known to cause stinging upon injection (Laursen T, et al, Basic clean Pharmacol Toxicol, 98 (2): 218-21 (2006)). For those drugs administered by the Subcutaneous (SC) or Intramuscular (IM) routes, where the drug solution stays at the site of administration for a relatively long time compared to the Intravenous (IV) route, where the formulation is rapidly diluted into the blood after administration, the stinging and irritation potential will be greater. For those formulations that are administered by direct intravenous infusion, it is desirable to monitor the total amount of buffer (and any other formulation ingredients). Particular care must be taken with the potassium ion used in the form of potassium phosphate, which causes cardiovascular effects in patients (Hollander-Rodriguez JC, et al, am. fam. Physician., 73 (2): 283-90 (2006)).

Buffers used in lyophilized formulations require additional considerations. Some buffers, such as sodium phosphate, crystallize out of the amorphous phase of the protein during freezing, resulting in a change in pH. Other common buffers such as acetic acid and imidazole may sublimate or evaporate during lyophilization, thereby changing the pH of the formulation during lyophilization or after reconstitution.

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

The presence of the pH buffering compound may be any suitable amount that will maintain the pH of the formulation at a predetermined level. In one embodiment, the pH buffered concentration is between 0.1mM and 500mM (1M). For example, contemplated pH buffers are at least 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 mM.

The pH buffers listed herein for buffering the formulation include, but are not limited to: organic acids, glycine, histidine, glutamic acid, succinate, phosphate, acetate, citrate, Tris, HEPES, and the following amino acids or amino acid mixtures: including but not limited to aspartic acid, histidine and glycine. In one embodiment of the invention, the buffer is citrate. Stabilizers and fillers

In one aspect of the pharmaceutical formulation of the present invention, a stabilizer (or combination of stabilizers) is added to avoid or reduce aggregation and chemical degradation caused by storage. A hazy or cloudy solution after reconstitution indicates precipitation or at least aggregation of the protein. The term "stabilizer" refers to an excipient that prevents aggregation or physical degradation in aqueous solution, including chemical degradation (e.g., autolysis, deamidation, oxidation, etc.). Contemplated stabilizers include, but are not limited to: sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, glycine, arginine hydrochloride, the following polyols: including polysaccharides such as dextran, starch, hydroxyethyl starch, cyclodextrin, N-methylpyrrolidone (N-methylpyrrolidinone), cellulose and hyaluronic acid, sodium chloride [ Carpenter et al, devilop.biol.standard 74: 225, (1991)]. In the formulations of the present invention, the stabilizer is included at a concentration of about 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, 900 or 1000 mM. In one embodiment of the invention, mannitol and trehalose are used as stabilizers.

If desired, the formulation may also include appropriate amounts of fillers and tonicity adjusting agents. Bulking agents include, for example, but are not limited to, mannitol, glycine, sucrose, polymers such as dextran, polyvinylpyrrolidone, carboxymethylcellulose, lactose, sorbitol, trehalose, xylitol. In one embodiment, the bulking agent is mannitol. The bulking agent is included at a concentration of about 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, 900, or 1000 mM. Surface active agent

Proteins have a higher tendency to interact with surfaces, which makes them susceptible to adsorption and denaturation at gas-liquid, bottle-liquid, and liquid-liquid (silicone oil) interfaces. This degradation pathway has been found to be inversely proportional to protein concentration and results in the formation of soluble and insoluble protein aggregates, or the loss of protein in solution by adsorption to the surface. In addition to adsorption on the surface of the container, surface-induced degradation is exacerbated by physical agitation as experienced during shipping and handling of the product.

Surfactants are commonly used in protein formulations to prevent surface-induced degradation. Surfactants are amphiphilic molecules that compete for protein at interfacial sites. The hydrophobic portion of the surfactant molecule occupies an interfacial location (e.g., gas/liquid), while the hydrophilic portion of the molecule remains directed toward the bulk of the solvent. At sufficient concentrations (usually around the critical micelle concentration of the detergent), the surface layer of surfactant molecules acts to prevent adsorption of protein molecules at the interface. Thus, surface-induced degradation is minimized. Surfactants contemplated herein include, but are not limited to: polysorbate 20 and polysorbate 80. The two differ only in the length of the fatty chain that confers the hydrophobic character of the molecule, C-12 and C-18 respectively. Thus, polysorbate 80 is more surface active and the critical micelle concentration is lower than polysorbate 20.

Detergents may also affect the thermodynamic conformational stability of a protein. Here, the effect of a given detergent excipient is protein specific. For example, polysorbates have been shown to decrease the stability of some proteins while increasing the stability of others. Detergent destabilization of proteins can be reasonably illustrated by the hydrophobic tail of the detergent molecule, which can participate in specific binding to partially or fully developed protein states. These types of interactions may result in a shift in the conformational equilibrium towards a more extended protein state (i.e. increased exposure of the hydrophobic portion of the protein molecule to compensate for binding to polysorbates). In addition, if the native state protein exhibits some hydrophobic surface, binding of the detergent to the native state will stabilize that conformation.

Another aspect of polysorbates is that they are themselves susceptible to oxidative degradation. Typically, as starting materials, they contain sufficient peroxide to cause oxidation of the side chains of residues on proteins, particularly methionine. The possibility of oxidative damage resulting from the addition of stabilizers underscores the point that the lowest effective concentration of excipients should be used in the formulation. For surfactants, the effective concentration for a particular protein will depend on the mechanism of stabilization.

Surfactants are also added in appropriate amounts to prevent aggregation during freezing and drying [ Chang, B, j.pharm.sci.85: 1325, (1996)]. Thus, examples of surfactants include, but are not limited to: anionic, cationic, nonionic, zwitterionic and amphoteric surfactants, including surfactants derived from naturally occurring amino acids. Anionic surfactants include, but are not limited to: sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate, chenodeoxycholic acid, N-lauroylsarcosine sodium salt, lithium lauryl sulfate, 1-octane sulfonate sodium salt, sodium cholate hydrate, sodium deoxycholate, and sodium glycerol deoxycholate. Cationic surfactants include, but are not limited to: benzalkonium chloride or benzethonium chloride, cetylpyridinium chloride monohydrate, and cetyltrimethylammonium bromide. Zwitterionic surfactants include, but are not limited to: CHAPS, CHAPSO, SB3-10, and SB 3-12. Nonionic surfactants include, but are not limited to: digitonin, Triton X-100, Triton X-114, Tween 20 and Tween 80. Surfactants also include, but are not limited to: lauromacrogol 400, polyoxyl 40 stearate, polyoxyl hydrogenated castor oil 10, 40, 50 and 60, glycerol monostearate, polysorbates 40, 60, 65 and 80, soy lecithin, and other phospholipids such as phosphatidyl choline Dioleate (DOPC), Dimyristoylphosphatidylglycerol (DMPG), Dimyristoylphosphatidylcholine (DMPC), and phosphatidyl glycerol Dioleate (DOPG); sucrose fatty acid ester, methylcellulose and carboxymethylcellulose. Thus, compositions containing these surfactants may further be provided, either as a single surfactant or as a mixture of different proportions. In one embodiment of the invention, tween 80 is the surfactant. In the formulations of the present invention, the surfactant is included at a concentration of about 0.01 to about 0.5 g/L. In the formulations provided, the surfactant concentration is 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 g/L. Salt (salt)

Salts are often added to increase the ionic strength of the formulation, which is important for protein solubility, physical stability, isotonicity. Salts can affect the physical stability of proteins in various ways. Ions stabilize the native state of a protein by binding charged residues on the surface of the protein. In addition, salts stabilize the denatured state of the protein by binding to a population of peptides along the backbone of the protein (-CONH-). Salts can also stabilize the native conformation of a protein by shielding electrostatic repulsive interactions between residues stabilized within one protein molecule. Salts in protein formulations also shield protein molecules from attractive electrostatic interactions that can lead to protein aggregation and insolubility. In the provided formulations, the salt concentration is between 0.1, 1, 10, 20, 30, 40, 50, 80, 100, 120, 150, 200, 300, and 500 mM. Other commonly used excipient ingredients amino acids

Amino acids have found a variety of uses in protein formulations as buffers, bulking agents, stabilizers and antioxidants. Thus, in one aspect, histidine and glutamic acid are used to buffer the protein formulation to a pH between 5.5-6.5 and 4.0-5.5, respectively. The pKa of the histidine imidazole group is 6.0 and the pKa of the glutamic acid side chain carboxyl group is 4.3, making these amino acids suitable for buffering at their respective pH ranges. Glutamic acid is particularly useful in this case. Histidine is commonly found in commercial protein formulations, and this amino acid provides a citrate replacement, and stinging upon citrate buffer injection is well known. Interestingly, histidine has also been reported to have a stabilizing effect against aggregation when used in high concentrations in liquid and lyophilized products (Chen B, et al, Pharm res., 20(12) (1952-60) (2003)). Histidine has also been observed by others to reduce the viscosity of formulations with high protein concentrations. However, in the same study, in a freeze-thaw study of the antibody in a stainless steel container, the authors observed increased polymerization and discoloration in histidine containing formulations. It should also be noted that histidine is subject to photooxidation in the presence of metal ions (Tomita M, et al, Biochemistry, 8 (12): 5149-60 (1969)). The use of methionine as an antioxidant formulation in formulations has shown promise, and has been found to be effective against a variety of oxidative stresses (Lam XM, et al, J Pharm ScL, 86 (11): 1250-5 (1997)).

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

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

Oxidation of protein residues occurs from a number of different causes. In addition to the addition of specific antioxidants, prevention of oxidative damage to proteins involves careful control of factors such as atmospheric oxygen, temperature, light, and chemical contamination throughout the manufacturing process and product storage. Thus, antioxidants for drugs contemplated for use in the present invention include, but are not limited to: reducing agents, oxygen scavengers/radical scavengers, or chelating agents. In one aspect, the antioxidants in the therapeutic protein formulations are water soluble and remain active throughout the shelf life of the product. The reducing agent and oxygen/radical scavenger function by ablating reactive oxygen species in the solution. Chelating agents such as EDTA are effective by binding trace metal contaminants that promote free radical formation. For example, EDTA is used to inhibit metal ion-catalyzed oxidation of cysteine residues in liquid formulations of acidic fibroblast growth factor.

In addition to the effectiveness of various excipients in preventing protein oxidation, concerns also need to be taken about the potential of the antioxidants themselves for causing other covalent or physical changes to the protein. For example, reducing agents can cause the cleavage of intramolecular disulfide bonds, resulting in shuffling of disulfide bonds. Ascorbic acid and EDTA have been shown to promote oxidation of methionine in some Proteins and Peptides in the presence of transition metal ions (Akers MJ and Defelippis MR.peptides and Proteins as particulate solutions. in: Pharmaceutical Formulation Development of Peptides and Proteins. Sven Frjar, Lars Hovgaard edition Pharmaceutical science. taylor and Francis, UK (1999)); fransson j.r.,/. pharm. sci.86 (9): 4046-1050 (1997); yin J, et al, Pharm res, 21 (12): 2377-83(2004)). Sodium thiosulfate has been reported to reduce light and temperature induced methionine oxidation levels in rhuMab HER 2; however, the formation of thiosulphate-protein adducts has also been reported in this report (Lam XM, Yang JY, et al, Jpharm Sci.86 (11): 1250-5 (1997)). The selection of a suitable antioxidant should be made according to the particular pressure and sensitivity of the protein. Antioxidants contemplated in certain aspects include, but are not limited to: reducing agents and oxygen scavengers/radical scavengers, EDTA, and sodium thiosulfate. Metal ion

Generally, transition metal ions in protein formulations are undesirable because they catalyze both physical and chemical degradation reactions of proteins. However, certain metal ions that can act as protein cofactors, as well as being capable of forming coordination complexes in protein suspension formulations (e.g., zinc suspensions of insulin), are included in the formulations. Recently, magnesium ion (10-120mM) has been proposed as an inhibitor of the iso-aspartic acid iso-constitution of aspartic acid (WO 2004039337).

Two examples of metal ions conferring increased stability or activity on proteins are human deoxyribonuclease (rhDNase, Pulmozyme)) And factor VIII. In one example of rhDNase, Ca⁺²Ions (not more than 100mM) increase enzyme stability through specific binding sites (Chen B, et al,/Pharm Sci, 88 (4): 477-82 (1999)). Indeed, removal of calcium ions from solution with EGTA results in increased deamidation and aggregation. However, this effect is only Ca⁺²Ion observed only; other divalent cations magnesium⁺²Manganese, manganese⁺²And zinc⁺²Destabilization of rhDNase was observed. Similar effects were found in the study of factor VIII. Ca⁺²And Sr⁺²The ions stabilize the protein, while others are Mg⁺²、Mn⁺²And Zn⁺²、Cu⁺²And Fe⁺²Destabilizing the enzyme (Fatouros, A., et al., int. J. pharm., 155, 121-131 (1997)). In another study of factor VIII, Al was found⁺³The aggregation rate in the presence of ions was significantly increased (Derrick TS, et al., pharm. Sci., 93 (10): 2549-57 (2004)). The authors noted that other excipients such as buffer salts tend to be coated with Al⁺³Ionic contamination and indicates the need to use excipients of the appropriate quality in formulating the product. Preservative

Preservatives are necessary in developing multi-purpose parenteral formulations involving multiple accesses from the same container. Its main function is to inhibit microbial growth and ensure sterility of the product throughout its shelf life or pharmaceutical product life. Commonly used preservatives include, but are not limited to: benzyl alcohol, phenol, m-cresol. While preservatives have a long history of use, developing protein formulations containing preservatives can be challenging. Preservatives almost always have a destabilizing effect (aggregation) on proteins, which has become a major factor limiting their use in multi-dose protein dosage forms ((Roy S, et al, J Pharm ScL, 94 (2): 382-96 (2005)).

Currently, most protein drugs are formulated for single use only. However, if multi-dose formulations are possible, they have the additional advantage of convenience for the patient, and improved marketability. As a good example, the development of preservative formulations of human growth hormone (hGH) has led to the marketing of more convenient, multiple use injection pen-type preparations. There are at least four such pen-type devices currently on the market that contain a preservative form of hGH. Norditropin(liquid, Novonide), Nutropin AQ(liquid, Gene technology Co.) and Genotropin (Freeze-drying-double Chamber, Pharmacia)&Upjohn) contains phenol, and Somatope(lilay corporation) contains m-cresol.

There are several aspects to be considered in developing preservative formulations. Effective preservative concentrations in pharmaceutical products must be optimized. This requires testing a given preservative for a range of concentrations that impart antimicrobial effectiveness within the formulation of the dosage form without affecting the stability of the protein. For example, three preservatives were successfully screened for the development of liquid formulations of interleukin 1 receptor (type I) using Differential Scanning Calorimetry (DSC). Preservatives are ranked according to their effect on stability at concentrations commonly used in commercial products (Remmele RL jr., et al, Pharm res., 15 (2): 200-8 (1998)).

The development of liquid dosage forms containing preservatives is more challenging than freeze-dried dosage forms. The freeze-dried product can be lyophilized without a preservative and reconstituted with a preservative-containing diluent at the time of reuse. This shortens the contact time of the preservative with the protein, significantly reducing the associated stability risks. For liquid formulations, the effectiveness and stability of the preservative must be maintained throughout the product shelf life (about 18-24 months). One important point to note is that preservative effectiveness must be demonstrated in the final formulation containing the active drug and all excipient ingredients.

Some preservatives can cause injection site reactions, another factor that needs to be considered in choosing a preservative. In Norditropin's clinical trials evaluated for preservatives and buffers, formulations containing phenol and benzyl alcohol were observed to have lower pain perception than formulations containing cresol (Kappelgaard AM, Horm Res.62 Suppl 3: 98-103 (2004)). Interestingly, among the commonly used preservatives, benzyl alcohol has an anaesthetic effect (Minogue SC, and Sun DA., AnesthAnalg., 100 (3): 683-6 (2005)). In various aspects, the use of preservatives provides benefits over any side effects. Preparation method

The present invention further contemplates methods for preparing pharmaceutical formulations.

The method further comprises one or more of the following steps: the stabilizing agent described herein is added to the mixture prior to lyophilization, and at least one agent selected from the group consisting of a bulking agent, an osmolality adjusting agent, and a surfactant is added to the mixture prior to lyophilization, wherein each agent is as described herein.

Standard reconstitution of lyophilized material is by adding back a volume (usually equivalent to the volume removed during lyophilization) of pure water for injection (WFI) or sterile water, although dilute solutions of antibacterial agents are sometimes used in the manufacture of parenterally administered drugs [ Chen, Drug Development and Industrial Pharmacy, 18: 1311-1354(1992)]. Thus, provided methods for preparing reconstituted rVWF compositions include adding a diluent to a lyophilized rVWF composition of the present invention.

The lyophilized material can be reconstituted as an aqueous solution. Aqueous carriers such as sterile water for injection, water with preservatives for multiple dose use, or water with appropriate amounts of surfactant (e.g., aqueous suspensions, where the mixture contains the active compound in admixture with excipients suitable for the manufacture of aqueous suspensions). In various embodiments, these excipients are suspending agents such as, but not limited to: sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents are a naturally occurring phospholipid such as, but not limited to: lecithin, or condensation products of alkylene oxides with stearic acid, such as but not limited to polyoxyethylene stearic acid, or condensation products of ethylene oxide with long chain aliphatic alcohols, such as but not limited to heptadecaethyl-phenoxyhexadecanol (heptadecadecaethyl-eneoxycetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and anhydrous hexitol, such as but not limited to polyethylene sorbitol monooleate. In various embodiments, the aqueous suspension also contains one or more preservatives, such as, but not limited to: ethyl benzoic acid, or n-propyl benzoic acid, p-hydroxybenzoic acid. Administration of drugs

In one aspect, for use of the composition in humans or test animals, the composition comprises one or more pharmaceutically acceptable carriers. As described below, the phrases "pharmaceutically" or "pharmacologically" acceptable means that the molecular entities and compositions are stable, inhibit protein degradation such as product polymerization and cleavage, and do not produce allergic, or other untoward reactions when administered using routes well known in the art. "pharmaceutically acceptable carrier" includes any and all clinically useful solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, including the agents disclosed above.

The pharmaceutical formulation is administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or by intracranial injection. The term "parenteral" as used herein includes subcutaneous injections, intravenous, intramuscular, intracisternal injection or infusion techniques. It is also contemplated that the specific site may be implanted by intravenous injection, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection and or surgery. Generally, the compositions are substantially free of pyrogens and other impurities that are detrimental to the recipient.

Single or multiple administrations of the composition are carried out at dosage levels and in a manner selected by the attending physician. For the prevention or treatment of disease, the appropriate dosage depends on the type, severity and course of the disease being treated as defined above, whether the drug is used for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the drug, and the discretion of the attendant physician. Reagent kit

As a supplemental aspect, the invention includes kits comprising one or more lyophilized compositions packaged in a manner that facilitates administration to a subject. In one embodiment, the kit comprises a pharmaceutical formulation (e.g., a composition comprising a therapeutic protein or peptide) as described herein, packaged in a container such as a sealed bottle or container, and a label affixed to the container or contained within the package that describes the particular use of the compound or composition in practicing the method. In one embodiment, the pharmaceutical formulation is packaged in a container such that the amount of headspace (e.g., the amount of air between the liquid formulation and the top of the container) within the container is very small. Preferably, the amount of headspace is negligible (i.e., almost none). In one embodiment, the kit comprises a first container containing a composition of a therapeutic protein or peptide and a second container containing a pharmaceutically acceptable reconstitution solution for the composition. In one aspect, the pharmaceutical formulation is packaged in unit dosage form. The kit may further comprise a device suitable for administering the pharmaceutical formulation according to a particular route of administration. Preferably, the kit contains a label to guide the use of the pharmaceutical formulation. Dosage form

The dosage regimen involved in the methods of treating the conditions described herein is determined by the attending physician, considering that various factors can affect the action of the drug, such as age, physical condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. By way of example, a typical dose of the recombinant VWF of the invention is about 50U/kg, which is equal to 500 μ g/kg.

In one aspect, administration of the formulation of the present invention is initiated by an initial bolus followed by a continuous infusion to maintain a therapeutic circulating level of the pharmaceutical product. As another example, the compounds of the present invention are administered in a single dose. Those of ordinary skill in the art will be readily able to optimize effective dosages and dosing regimens as determined by good medical practice and the clinical condition of the individual patient. The frequency of administration depends on the pharmacokinetic parameters of the agent and the route of administration. The optimal pharmaceutical dosage form is determined by one skilled in the art based on the route of administration and the desired dosage. See, for example, Remington's pharmaceutical sciences, 18th Ed, (1990, Mack Publishing co., Easton, PA 18042) at pages 1435 to 1712, the disclosure of which is incorporated herein by reference. These dosage forms affect the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered agent. Depending on the route of administration, suitable doses are calculated in terms of body weight, body surface area or organ size. The appropriate dose can be determined by using established analytical methods for determining blood levels in combination with appropriate dose response data. The final dosage regimen will be determined by the attending physician, considering that various factors will influence the action of the drug, such as the specific activity of the drug, the severity of the injury and the responsiveness of the patient, age, physical condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Further information will emerge as the study progresses regarding appropriate dosage levels and duration of treatment for different diseases and conditions.

The following examples are intended to be merely illustrative of specific embodiments of the invention and are not intended to be limiting. Example 1 shaking experiment

To determine the amount of rVWF precipitation in different formulations, the extent of rVWF aggregation after vigorous shaking was tested under various conditions.

As shown in table 1, various rVWF formulations were evaluated in 20mM citrate buffer, pH 7.3. Of shaking experimentsThe aim is to simulate mechanical pressure conditions. Each formulation was prepared in 1-2ml portions on a laboratory shaker at 1200rpm for 10 minutes. TABLE 1

Evaluation of the visible VWF aggregation was performed according to the protocol shown below. In most cases, "visible aggregates" are colloidal fibers varying in size from about 100 nanometers to 1-2 centimeters. Scheme(s)

Granules
		A	Particle-free
B	A number of particles, rarely seen (dots)
		B1	More grains, less visible (dots)
C	Several particles, easy to see (fiber)
		D	More particles, easy to see (fiber)
E	Visible particle (> 1mm fibre)
		E1	Fluffy white precipitate (floating on the surface)
E2	Jellyfish-like

The results of the shaking experiments are shown in Table 2 below. TABLE 2

In summary, the shaking experiments described above show that the formulation containing tween 80 and mannitol provides the best results (i.e. minimal aggregation). Example 2 Freeze-thaw experiments

Freezing and thawing testThe purpose of the experiment was to assess the effect of stress caused by repeated freeze-thawing. In addition to the formulations in the above shaking experiments (see table 1), the following formulations (tables 3 and 4) were also evaluated. TABLE 3TABLE 4

All formulations were frozen in a freezer at-20 ℃ for about 1 hour and then thawed at room temperature. The results are given in Table 5 below. TABLE 5

As indicated above, trehalose provided the best results (i.e. minimal amount of aggregation). Example 3 Freeze drying experiments

The purpose of the freeze-drying experiment was to evaluate the ability of various formulations to allow the formation of a lyophilized cake that could dissolve in less than 10 minutes and become a clear solution. Accelerated stability studies have also been performed to demonstrate no significant loss of biological activity.

The formulations shown in table 6 below were lyophilized using a nitrogen lyophilizer TS20002 according to the manufacturer's instructions. The total time for lyophilization was about 72 hours. Each of the following formulations also contained 20g/L mannitol and 0.1g/L Tween 80.

The results of the lyophilization experiments are shown in Table 7 below.

As shown in the table above, the combination of citrate or HEPES with amino acids provided the clearest solution.

To evaluate the stability of reconstituted lyophilized rVWF, VWF: Ag and VWF: RCo tests were performed. Ag corresponds to the use of a polyclonal anti-VWF antibodyThe VWF-specific ELISA of the bodies can detect the amount of VWF, whereas VWF: RCo corresponds to the amount of VWF that leads to the aggregation of the stabilized platelets in the presence of risticidin. Samples were stored at 40 ℃. Assuming that the Arrhenius equation applies, the stability at 40 ℃ for one month is equivalent to about one year at 4 ℃. The results of the stability experiments are shown in tables 8 and 9 below. TABLE 8TABLE 9

The standard deviation of the ELISA was in the range of 10-20%. The above results show that all tested formulations provided good stability at 40 ℃ over a period of 8 weeks.

Supplementary stability experiments were performed in which different amino acids (e.g.glycine, lysine or histidine at 15mM or 20mM) were used in the formulation and citrate buffer was varied (e.g.15, 20 or 25 mM). As described above, the stability of rVWF was monitored using the VWF: RCo activity assay. Even after 13 months, no significant difference in VWF to RCo activity values was observed for the rVWF stability samples stored at 40 ℃. The significance of the measurements was tested by t-test. The medium precision of the analytical method is obtained by calculating the Coefficient of variation (coeffient of Variance). The Coefficient of Variation (CV) was below 20% in all series of stability data, meeting the validation criteria of CV < 20%. From the above results, it can be concluded that rVWF is stable in all citrate buffer systems tested, regardless of buffer molarity and added amino acids. rVWF remains stable for at least 13 months even when stored at 40 ℃. Potency assay using VWF: RCo activity assay showed good medium precision with CV values below 20%.

Thus, according to the data presented herein, a formulation of rVWF is presented comprising 15mM citrate (trisodium citrate dihydrate), 15mM glycine, 10g/L trehalose, 20g/L mannitol, 0.1g/L tween 80, pH 7.3. Example 4 Long term stability

Accelerated and Long term stability test

A study was conducted to evaluate the stability of the final pharmaceutical product (FDP) of rVWF to storage under recommended and elevated temperature storage conditions. The resulting data from high temperature storage conditions can ensure that temperature deviations do not affect the quality of rVWF FDP and can be used to extrapolate acceptable failure conditions for materials without real-time, real-condition stability data.

The current specification is < 3.0% residual moisture (determined using Karl Fischer method). The lot numbers rvwf #4FC, rvwf #5FC, rvwf #6FC and rvwf #7FC released moisture contents of 1.2%, 1.3%, 1.2% and 1.5%, respectively. From past experience with other products using similar vial and stopper configurations, it is expected that a batch of rVWF that can release about 1.3% of any residual moisture will meet the specification limit of ≦ 3.0% at the end of the proposed shelf life (i.e., 24 months at the intended storage temperature of 5 ℃ ± 3 ℃).

The four batches of rVWF FDP produced were used to conduct long-term stability studies at the recommended storage conditions (i.e., 5 ℃ ± 3 ℃) and high temperatures (i.e., 40 ℃ ± 2 ℃). These studies provide sufficient data to compare stable performance of individual clinical batches.

The experimental protocol for stability, including the stability indication analysis and specification of stability acceptance criteria, can be found in table 10, which also contains information regarding the lot number of rVWF FDP evaluated in the stability study. Watch 10

Summary and discussion of Total stability (24 months)

The stability data for the rVWF FDP listed include the following data:

1. long-term study data for batch rVWF #1FC at 5 ℃ ± 3 ℃ for 24 months (complete test) and intermediate data at 30 ℃ ± 2 ℃ for 6 months (complete test);

2. data for batch rVWF #2FC at 5 ℃ ± 3 ℃ for 6 months (complete test);

3. long-term study data for batch rVWF #3FC at 2-8 ℃ for 24 months (full test), data at 30 ℃ ± 2 ℃ for 6 months, and data at 40 ℃ ± 2 ℃ for 3 months (full test);

4. stability data for batch rVWF #4FC at 5 ℃ ± 3 ℃ for 24 months and data at 40 ℃ ± 2 ℃ for 9 months;

5. stability data for batch rVWF #5FC at 5 ℃ ± 3 ℃ for 24 months and data at 40 ℃ ± 2 ℃ for 9 months;

6. stability data for batch rVWF #6FC at 5 ℃ ± 3 ℃ for 12 months and data at 40 ℃ ± 2 ℃ for 9 months; and

7. stability data for batch number rVWF #7FC at 5 ℃ ± 3 ℃ for 12 months and data at 40 ℃ ± 2 ℃ for 9 months

The residual moisture observed in lot numbers rVWFF #4FC, rVWFF #5FC, rVWFF #6FC, and rVWFF #7FC remained well below the qualification standard ≦ 3% with no impact on functional activity (VWF: RCo). For batches produced to be suitable for non-clinical and clinical studies, no observable changes were seen in the qualitative analytical techniques (i.e., appearance, SDS-PAGE electrophoretic analysis, etc.). Similarly, no trend was seen for decreasing stability in the number of VWF multimers observed during total protein analysis, VWF: Ag analysis, or storage.

The ratio of VWF: RCo activity to VWF: Ag activity, and the variation in VWF: RCo data, present in lot numbers rVWF #1FC, rVWF #2FC, and rVWF #3FC, may be the result of variations in the test method, in fact, the individual VWF: RCo stability test results consist of single-fix data of a single stability sample, and/or data from non-european pharmacopoeia-consistent analytical methods. After the test method was modified to the test method according to the european pharmacopoeia, all test time points of non-clinical batches were tested using the original and new assay methods.

Mass productionThe rVWF FDP of (a) shows stability characteristics similar to those of experimentally produced rVWF FDP. These batches of rVWF FDP maintained VWF: RCo activity at least after 24 months of storage at 5 ℃ ± 3 ℃. No change in VWF multimer pattern was seen in the large scale batches of stability samples currently tested, even when stored at 30 ℃ ± 2 ℃ for 6 months or 40 ℃ ± 2 ℃ for 9 months. Table 11 lists the results of the VWF: RCo, VWF: Ag and VWF multimer patterns for the rVWF #4FC, rVWF #5FC, rVWF #6FC and rVWF #7FC stored at a conditional pressure of 40 ℃. + -. 2 ℃. The results show a stability of 9 months of storage at elevated temperature, which can be extrapolated to a shelf life of more than 3 years at ambient temperature or even longer under refrigerated conditions. TABLE 11

Covariance analysis (ANCOVA analysis) indicated that the difference in the slopes of the regression lines (lot numbers rVWFF #4FC, rVWFF #5FC, rVWFF #6FC and rVWFF #7FC stored at 5 ℃ ± 3 ℃) was not significant (p ═ 0.906), allowing VWF: RCo activity data to be merged as described in ICH Q1A (R2). The trend line height difference for individual batches was also not significant. As shown in fig. 1, extrapolating the worse case slope of the merge shows that the confidence interval is well within the acceptance criteria for the lowest value of 24 months. By 51 months, the under-lying interval of the mean curve dropped to 80% of the initial activity (80% is also the maximum difference between predicted and annotated potency given by human von willebrand factor in the european pharmacopoeia). The combined worse case slopes show a monthly reduction of 0.0344U VWF to RCo. This comparison shows that the stability characteristics of rVWF FDP, in particular VWF: RCo activity, are not changed due to production process variations. The above extrapolation supports the possibility to extend the temporary shelf life of rVWF FDP to 24 months at the recommended storage temperature.

The transfer of moisture from the stopper to the lyophilized product is dependent on the stopper material and is affected by residual moisture after sterilization of the stopper, the humidity of the sample stored, and the inherent moisture transfer rate of the stopper. As shown in fig. 2, the residual moisture of the lot numbers rVWFF #4FC, rVWFF #5FC, rVWFF #6FC and rVWFF #7FC stored at 5 ℃ ± 3 ℃ was similar (the difference in comparison between slopes was not significant, p ═ 0.734). The batch stored for 9 months at elevated temperature 40 ℃ ± 2 ℃ showed a corresponding increase in residual moisture (fig. 3). Here, ANCOVA analysis showed that the slope difference at the regression line was comparable (p ═ 0.546). Figure 3 shows an extrapolation of the worse case combined slope to 24 months.

These sufficient data support that lot numbers rVWFF #6FC and rVWFF #7FC can be used within the 24 month shelf life time if stored at 5 ℃ ± 3 ℃.

Recommended storage conditions and shelf life

Recommended storage conditions for rVWF FDP are 5 ℃ ± 3 ℃. Therefore, it is recommended that the tentative shelf life of rVWF FDP is 24 months if stored under recommended storage conditions. The shelf life of rVWF FDP may be further extended based on the supplemental data obtained with longer shelf life.

Claims (16)

1. A stable lyophilized pharmaceutical formulation of recombinant von willebrand factor (rVWF) comprising: (a) rVWF; (b) one or more buffering agents; (c) more than one amino acid; (d) one or more stabilizers; and (e) one or more surfactants;

the rVWF comprises a polypeptide selected from the group consisting of:

a) the amino acid sequence shown as SEQ ID No. 3;

b) a biologically active analog, fragment or variant of said a);

c) a polypeptide encoded by the polynucleotide shown in SEQ ID No. 1;

d) a biologically active analog, fragment or variant of said c); and

e) a polypeptide encoded by a polynucleotide that hybridizes under moderately stringent hybridization conditions to the polynucleotide of SEQ ID No. 1, wherein said moderately stringent hybridization conditions refer to the following conditions: hybridization in 50% formamide at 42 ℃ and washing in 0.1 XSSC, 0.1% SDS at 60 ℃;

the buffer comprises a pH buffer, the buffer concentration is in the range of 1mM to 100mM, and the pH is in the range of 6.0 to 8.0;

the concentration of the amino acid is 1mM to 100 mM;

the concentration of the stabilizer is 1g/L to 50 g/L; and is

The concentration of the surfactant is 0.05g/L to 0.5 g/L.

2. The formulation of claim 1, wherein the rVWF comprises the amino acid sequence set forth in SEQ ID No. 3.

3. The formulation of claim 1, wherein the buffering agent is selected from the group consisting of citrate, glycine, histidine, HEPES, Tris, and combinations of these agents.

4. The formulation of claim 3, wherein the buffer is citrate.

5. The formulation of claim 1, wherein the pH is in the range of 6.5 to 7.5.

6. The formulation of claim 5, wherein the pH is 7.3.

7. The formulation of claim 1, wherein the buffer is citrate and the pH is 7.3.

8. The formulation of claim 1, wherein the amino acid is selected from the group consisting of glycine, histidine, proline, serine, alanine, and arginine.

9. The formulation of claim 8, wherein the amino acid is in a concentration range of 5mM to 50 mM.

10. The formulation of claim 9, wherein the amino acid is glycine at a concentration of 15 mM.

11. The formulation of claim 1, wherein the rVWF comprises the amino acid sequence set forth in SEQ ID No: 3; the buffer is citrate and has a pH of 7.3; and the amino acid is glycine at a concentration of 15 mM.

12. The formulation of claim 1, wherein the one or more stabilizing agents are selected from the group consisting of mannitol, lactose, sorbitol, xylitol, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, and combinations of these stabilizing agents.

13. The formulation of claim 12, wherein the stabilizing agent is trehalose at a concentration of 10g/L mM and mannitol at a concentration of 20 g/L.

14. The formulation of claim 1, wherein said surfactant is selected from the group consisting of digitonin, Triton X-100, Triton X-114, tween-20, tween-80, and combinations of these surfactants.

15. The formulation of claim 14, wherein the surfactant is 0.01g/L tween-80.

16. The formulation of claim 1, wherein the rVWF comprises the amino acid sequence set forth in SEQ ID No: 3; the buffer is citrate at a concentration of 15mM and a pH of 7.3; the amino acid is glycine with a concentration of 15 mM; the stabilizer is trehalose with the concentration of 10g/L and mannitol with the concentration of 20 g/L; and the surfactant is tween-80 at a concentration of 0.1 g/L.