Self-buffering protein formulations

Abstract

To provide self-buffering pharmaceutical protein formulations that are suitable for veterinary and human medical use.SOLUTION: Self-buffering protein formulations are substantially free of other buffering agents, stably maintain pH for the extended time periods involved in the distribution and storage of pharmaceutical proteins for veterinary and human medical use. The invention further provides methods for designing, making, and using the formulation. In addition to other advantages, the formulations avoid the disadvantages associated with the buffering agents conventionally used in current formulations of proteins for pharmaceutical use. The invention in these and other respects can be productively applied to a wide variety of proteins and is particularly useful for making and using self-buffering formulations of pharmaceutical proteins for veterinary and medical use, especially, for the treatment of diseases in human subjects.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to protein formulations, especially pharmaceutical proteins. In particular, the present invention relates to self-buffering biopharmaceutical protein compositions and methods for designing, producing and using said compositions. Furthermore, the present invention relates to pharmaceutical protein compositions for animal (veterinary) and / or human medical use and related methods. [Background technology]

[0002] 2. Background of the Invention Many aspects of pharmaceutical production and formulation processes are pH sensitive: maintaining the correct pH of the final drug product is critical to its stability, efficacy and shelf life, and pH is an important consideration in the design of a formulation for acceptable, safe and effective administration.

[0003] To maintain pH, one or more buffering agents are used in pharmaceutical processing and formulation. A wide range of buffering agents are available for pharmaceutical use. The buffer or buffers for a given application must be effective at the desired pH. These buffers must provide sufficient buffering capacity to maintain the desired pH for as long as necessary. A good buffer for a pharmaceutical composition must also meet many other criteria. The buffer must be reasonably soluble. The buffer must not form harmful complexes with metal ions, be toxic, or be excessively permeable, soluble, or absorbed on membranes or other surfaces. The buffer must not interact with other components of the composition in any way that reduces its availability or effectiveness. The buffer must be stable and effective in maintaining pH over the range of conditions to which it is likely to be exposed during the formulation and storage of the product. It must not be adversely affected by oxidation or other reactions occurring in its environment, such as reactions occurring in the processing of the composition to which it provides buffering. If carried over or incorporated into the final product, the buffer must be safe for administration, compatible with the other ingredients of the composition over the shelf life of the product, and acceptable for administration to the end user.

[0004] Although there are many buffers in general use, the number of buffers suitable for biological applications is limited, and of these, only a few are acceptable for pharmaceutical processes and formulations. As a result, it is often difficult to find a buffer that is not only effective in maintaining pH for a given pharmaceutical process, formulation, or product, but also meets all other requirements.

[0005] The challenge of finding suitable buffers for pharmaceutical applications is particularly acute for pharmaceutical proteins. Protein conformation and activity are highly pH dependent. Proteins are susceptible to a variety of pH-sensitive reactions that are detrimental to their efficacy (reactions that typically go far beyond those that affect small molecule drugs). For example, asparagine and glutamine side chain amides deamidate at low pH (below 4.0) and also at neutral or high pH (above 6.0), to name just a few prominent examples. Aspartic acid residues promote hydrolysis of adjacent peptide bonds at low pH. Disulfide bond stability and configuration are highly pH dependent, especially in the presence of thiols. Protein solubility, aggregation, aggregation, precipitation, and fibrillation are strongly pH dependent. Crystal habits, important for some pharmaceutical formulations, are also strongly pH dependent. Furthermore, pH is also an important factor in the surface absorption of many pharmaceutical peptides and proteins.

[0006] Buffers that catalyze reactions that inactivate and / or degrade one or more other components also cannot be used in pharmaceutical formulations. Buffers for pharmaceutical use not only have the buffering capacity required to maintain the correct pH, but also must not buffer so strongly that their administration adversely disrupts the physiological pH of the subject. Buffers for pharmaceutical formulations must also be compatible with the generally complex formulation process. For example, buffers that sublimate or evaporate (e.g., acetate and imidazole) usually cannot be relied upon to maintain pH during lyophilization or of reconstituted lyophilized products. Other buffers that crystallize the amorphous phase of the protein, such as sodium phosphate, cannot be relied upon to maintain pH in processes that require freezing.

[0007] Buffers used to maintain pH in pharmaceutical end products must also be not only effective in maintaining pH, but also safe and tolerable for administration to a subject. For example, some otherwise useful buffers, such as low or high citrate and high acetate, are undesirably painful when administered parenterally.

[0008] Some buffers have been found to be useful in formulating pharmaceutical proteins (e.g., acetate, succinate, citrate, histidine (imidazole), phosphate, and Tris). All of these have undesirable limitations and disadvantages. They also all have the inherent disadvantages of being additional components in the formulation, complicating the formulation process and introducing the risk of adversely affecting other components, stability, shelf life, and end-user acceptability. Summary of the Invention [Problem to be solved by the invention]

[0009] Thus, there is a need for further and improved methods of maintaining the pH of pharmaceutical production and formulations and pharmaceutical compositions, particularly biopharmaceutical protein production and formulations and biopharmaceutical protein compositions. [Means for solving the problem]

[0010] Summary of the Invention Thus, among the various objects and aspects provided by the present invention in certain preferred embodiments are protein formulations comprising proteins, particularly pharma- ceutically acceptable formulations comprising pharmaceutical proteins, which are buffered by the protein itself without the need for additional buffering agents to maintain a desired pH, and protein formulations in which the protein is substantially the only buffering agent (i.e., other components, if any, do not substantially act as buffering agents in the formulation).

[0011] In this regard and others, one of the various objects and aspects provided by the present invention in certain preferred embodiments is a self-buffering formulation of a protein, particularly a pharmaceutical protein formulation characterized in that the concentration of the formulated protein provides a desired buffering capacity.

[0012] Among the various objects and aspects provided by the present invention in certain particularly preferred embodiments are self-buffering protein formulations, particularly pharmaceutical protein formulations, having a total salt concentration of less than 150 mM.

[0013] Among the various objects and aspects provided by the present invention in certain particularly preferred embodiments are self-buffering protein formulations, particularly pharmaceutical protein formulations further comprising one or more polyols and / or one or more surfactants.

[0014] Furthermore, one of the various objects and aspects provided by the present invention in certain particularly preferred embodiments is a self-buffering formulation comprising a protein, particularly a pharmaceutical protein, wherein the total salt concentration in the formulation is less than 150 mM, and further comprising one or more excipients, such as, but not limited to, a pharma- ceutically acceptable salt; an osmotic balancing agent (isotonicity agent); a surfactant, a polyol, an antioxidant; an antibiotic; an antifungal agent; a bulking agent; a lyoprotectant; an antifoaming agent; a chelating agent; a preservative; a coloring agent; and an analgesic agent.

[0015] Among the various objects and aspects provided by the present invention in certain preferred embodiments are self-buffering protein formulations, particularly pharmaceutical protein formulations that contain, in addition to the protein, one or more other pharma- ceutical active agents.

[0016] Various additional aspects and embodiments of the invention are specifically described in the following numbered paragraphs. The invention is described by reference to each of the items described in the paragraphs, individually and / or jointly in any combination. Applicant expressly reserves the right to assert claims based on any such combination.

[0017] 1. A composition according to any of the following compositions, wherein the composition has been approved for its pharmaceutical use by a national or international authority authorized by law to grant such approval, preferably the European Medicines Agency, the Ministry of Health, Labor and Welfare of Japan, the National Medical Products Administration of China, the United States Food and Drug Administration, or a sub-agency within said authority, particularly preferably the United States Food and Drug Administration, or a sub-agency within said authority.

[0018] 2. Any of the above or below compositions, wherein said composition is manufactured in accordance with good manufacturing practices applicable to the manufacture of pharmaceuticals for human use.

[0019] 3. Any of the above or below compositions comprising a protein, the protein having a buffering capacity per unit volume and per pH unit equal to or greater than the buffering capacity of a sodium acetate buffer of about 2.0 or 3.0 or 4.0 or 5.0 or 6.50 or 8.00 or 10.0 or 15.0 or 20.0 or 30.0 or 40.0 or 50.0 or 75.0 or 100 or 125 or 150 or 200 or 250 or 300 or 350 or 400 or 500 mM, particularly preferably at least 2.0 mM, very particularly preferably at least 3.0 mM, even very particularly preferably at least 4.0 mM or at least 5.0 mM, very particularly preferably at least 7.5 mM, particularly preferably at least 10 mM, preferably at least 20 mM, in pure water over the pH range of 5.0-4.0 or 5.0-5.5, which buffering capacity is preferably measured by the method described in Examples 1 and 2.

[0020] 4. Any of the above or below compositions, wherein the buffer capacity of the composition per unit volume and per pH unit, excluding the buffer capacity of the protein, is equal to or smaller than the buffer capacity of 1.0 or 1.5 or 2.0 or 3.0 or 4.0 or 5.0 mM sodium acetate buffer in pure water over the pH range 4.0-5.0 or pH 5.0-5.5, particularly preferably less than a buffer capacity of 1.0 mM, even more particularly preferably less than a buffer capacity of 2.0 mM, very particularly preferably less than a buffer capacity of 2.5 mM, particularly preferably less than a buffer capacity of 3.0 mM, preferably less than a buffer capacity of 5.0 mM, said buffer capacity being preferably measured by the method described in Examples 1 and 2.

[0021] 5. Any of the above or below compositions comprising a protein, wherein the buffering capacity of the protein is at least about 1.00 or 1.50 or 1.63 or 2.00 or 3.00 or 4.00 or 5.00 or 6.50 or 8.00 or 10.0 or 15.0 or 20.0 or 30.0 or 40.0 or 50.0 or 75.0 or 100 or 125 or 150 or 200 or 250 or 300 or 350 or 400 or 500 or 700 or 1,000 mEq / liter (l) / pH unit over a range of ±1 pH unit from the pH of the composition, preferably at least about 1.00, particularly preferably 1.50, very particularly preferably 1.63, even more particularly preferably 2.00, even more very particularly preferably 3.00, even more very particularly preferably 5.0, very particularly preferably 10.0, and especially preferably 20.0 The composition, in mEq / l / pH units.

[0022] 6. Any of the above or below compositions comprising a protein, wherein the buffer capacity of the composition, excluding the protein, per unit volume and per pH unit over a range of ±1 pH unit from the pH of the composition, is equal to or less than the buffer capacity of 0.50 or 1.00 or 1.50 or 2.00 or 3.00 or 4.00 or 5.00 or 6.50 or 8.00 or 10.0 or 20.0 or 25.0 mM sodium acetate buffer in pure water over the pH range 5.0-4.0 or pH 5.0-5.5, wherein this buffer capacity is particularly preferably measured according to Example 1 and / or 2.

[0023] 7. Any of the above or below compositions, wherein the protein provides a buffering capacity of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of the buffering capacity of the composition over a range of ±1 pH unit from the desired pH, preferably at least about 75%, particularly preferably at least about 85%, very particularly preferably at least about 90%, even very particularly preferably at least about 95%, and even very very particularly preferably at least about 99% of the buffering capacity of the composition.

[0024] 8. Any of the above or below compositions, wherein the concentration of the protein is about 20 to 400, or about 20 to 300, or about 20 to 250, or about 20 to 200, or about 20 to 150 mg / ml, preferably about 20 to 400 mg / ml, particularly preferably about 20 to 250, and very particularly preferably about 20 to 150 mg / ml.

[0025] 9. Any of the above or below compositions, wherein the pH maintained by the buffering action of the protein is about 3.5 to 8.0, or about 4.0 to 6.0, or about 4.0 to 5.5, or about 4.0 to 5.0, preferably about 3.5 to 8.0, and most preferably about 4.0 to 5.5.

[0026] 10. Any of the above or below compositions, wherein the salt concentration is less than 150 mM or 125 mM or 100 mM or 75 mM or 50 mM or 25 mM, preferably 150 mM, particularly preferably 125 mM, very preferably 100 mM, even more particularly preferably 75 mM, particularly preferably 50 mM, preferably less than 25 mM.

[0027] 11. Any of the above or below compositions, further comprising one or more pharma- ceutical acceptable salts; polyols; surfactants; osmotic balancing agents; isotonicity agents; antioxidants; antibiotics; antimycotics; bulking agents; lyoprotectants; antifoaming agents; chelating agents; preservatives; coloring agents; analgesics; or additional agents.

[0028] 12. Any of the above or below compositions comprising one or more hypotonic, isotonic or hypertonic, preferably approximately isotonic and particularly preferably an isotonic amount of a pharma- ceutically acceptable polyol, very preferably any one or more of sorbitol, mannitol, sucrose, trehalose or glycerol, particularly very preferably about 5% sorbitol, 5% mannitol, 9% sucrose, 9% trehalose or 2.5% glycerol, and more particularly in this regard 5% sorbitol, 5% mannitol, 9% sucrose, 9% trehalose or 2.5% glycerol.

[0029] 13. Any of the above or below compositions, further comprising a surfactant, preferably one or more of polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan, polyethoxylates, and poloxamer 188, particularly preferably polysorbate 20 or polysorbate 80, preferably about 0.001-0.1% polysorbate 20 or polysorbate 80, even more preferably about 0.002-0.02% polysorbate 20 or polysorbate 80, particularly 0.002-0.02% polysorbate 20 or polysorbate 80.

[0030] 14. Any of the above or below compositions, wherein the protein is a pharmaceutical agent and said composition is a sterile preparation thereof suitable for treatment of a non-human or human subject.

[0031] 15. Any of the above or below compositions, wherein the protein is a pharmaceutical agent effective in treating a disease, and the composition is a sterile preparation of the protein suitable for administration to a subject to treat the disease.

[0032] 16. Any of the above or below compositions, wherein the protein does not induce a significant adverse antigenic response after administration to a subject.

[0033] 17. Any of the above or below compositions, wherein the protein does not induce a significant deleterious immune response after administration to a subject.

[0034] 18. Any of the above or below compositions, wherein the protein is a human protein.

[0035] 19. Any of the above or below compositions, wherein the protein is a humanized protein.

[0036] 20. Any of the above or below methods, wherein the protein is an antibody, preferably an IgA, IgD, IgE, IgG or IgM antibody, particularly preferably an IgG antibody, very particularly preferably an IgG1, IgG2, IgG3 or IgG4 antibody, in particular an IgG2 antibody.

[0037] 21. Any of the above or below compositions, wherein the protein is a Fab fragment, a Fab 2 Fragment, Fab 3 fragment, Fc fragment, scFv fragment, bis-scFv(s) fragment, minibody, dimer, trimer, tetrabody, VhH domain, V-NAR domain, V H Domain, V LThe above composition which is a domain, a camelid Ig, an Ig NAR, or a peptibody, or a variant, derivative or modification of any of the above antibodies.

[0038] 22. Any of the above or below compositions, wherein the protein comprises an Fc fragment or part thereof, or a derivative or variant of an Fc fragment or part thereof.

[0039] 23. Any of the above or below compositions, wherein the protein comprises a first binding portion of a pair of cognate binding moieties, wherein the first portion specifically binds to the second portion.

[0040] 24. Any of the above or below compositions, wherein the protein comprises (a) an Fc fragment or portion thereof, or a derivative or variant of an Fc fragment or portion thereof, and (b) a first binding portion of a pair of cognate binding moieties.

[0041] 25. The composition of any of claims 1, 5, 7, 9, 11, 13, or 14, wherein the protein is selected from the group consisting of proteins that specifically bind to one or more of CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factors, fibroblast growth factors, transforming growth factors (TGFs), insulin-like growth factors, bone morphogenetic factors, insulin and insulin-related proteins, aggregation and aggregation-related proteins, colony stimulating factors (CSFs), other blood proteins and serum proteins; blood group antigens; receptors, receptor-related proteins, growth hormone receptors, T-cell receptors; neurotrophic factors, neurotrophins, relaxins, interferons, interleukins, viral antigens, lipoproteins, integrins, rheumatoid factors, immunotoxins, surface membrane proteins, transport proteins, homing receptors, addressins, regulatory proteins, and immunoadhesins.

[0042] 26. Any of the above or below compositions, wherein the protein is selected from the group consisting of OPGL-specific binding protein, myostatin-specific binding protein, IL-4 receptor-specific binding protein, IL1-R1-specific binding protein, Ang2-specific binding protein, NGF-specific binding protein, CD22-specific binding protein, IGF-1 receptor-specific binding protein, B7RP-1-specific binding protein, IFNγ-specific binding protein, TALL-1-specific binding protein, stem cell factor, Flt-3 ligand, and IL-17 receptor.

[0043] 27. Any of the above or below compositions, wherein the protein is CD3, CD4, CD8, CD19, CD20, CD34; HER2, HER3, HER4, EGF receptor; LFA-1, Mol, p150, 95, VLA-4, ICAM-1, VCAM, αv / β3 integrin; vascular endothelial growth factor ("VEGF"); growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, Mullerian inhibitory substance, human macrophage inflammatory protein (MIP-1-α), erythropoietin (EPO), NGF-β, platelet derived growth factor (PDGF), aFGF, bFGF, epidermal growth factor (EGF), TGF-α, TGF-β1, TGF-β2, TGF-β3, T GF-β4, TGF-β5, IGF-I, IGF-II, des(1-3)-IGF-I (brain IGF-I), insulin, insulin A chain, insulin B chain, proinsulin, insulin-like growth factor binding proteins; for example, factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1-antitrypsin, plasminogen activators (e.g., urokinase) and tissue plasminogen activator ("t-PA"), bombazine, thrombin, and thrombopoietin, among others; M-CSF, GM-CSF, G-CSF, albumin, IgE, flk2 / flt3 receptor, obesity (OB) receptor, brain-derived neurotrophic factor (BDNF) (NT-3, NT-4, NT-5, NT-6); The above composition is selected from the group consisting of proteins that specifically bind to one or more of relaxin A chain, relaxin B chain, prorelaxin; interferon-alpha, -beta, and -gamma; IL-1 to IL-10; AIDS envelope viral antigen; calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor-alpha and -beta, enkephalinase, RANTES, mouse gonadotropin-related peptide, deoxyribonuclease (Dnase), inhibin, and activin; protein A or D, bone morphogenetic protein (BMP), superoxide dismutase, and decay accelerating factor (DAF).

[0044] 28. Any of the above or below compositions, wherein the protein is Actimmune (interferon-gamma-1b), Activase (alteplase), Aldurazme (laronidase), Amevive (alefacept), Avonex (interferon beta-1a), BeneFIX (nonacog alfa), Beromun (tasonermin), Beatseron (interferon-beta-1b), BEXXAR (tositumomab), Tev-Tropin (somatropin), Bioclate or RECOMBINATE (recombinant), CEREZME (imiglucerase), ENBREL (etanercept), Eprex (epoetin alfa), EPOGEN / Procit (epoetin alfa), FABRAZYME (agalsidase beta), Fasturtec / Elitek ELITEK (rasburicase), FORTEO (teriparatide), GENOTROPIN (somatropin), GlucaGen (glucagon), Glucagon (glucagon, rDNA derived), GONAL-F (follitropin alfa), KOGENATE FS (ostocog alfa), HERCEPTIN (trastuzumab), HUMATROPE (somatropin), HUMIRA (adalimumab), insulin in solution, INFERGEN® (interferon alfacon-1), KINERET® (anakinra), Kogenate FS (Antihemophilic Factor), LEUKIN (Sargramostim Recombinant Human Granulocyte-Macrophage Colony-Stimulating Factor (rhuGM-CSF)), CAMPATH (Alemtuzumab), RITUXAN® (Rituximab), TNKase (Tenecteplase), MYLOTARG (Gemtuzumab Ozogamicin), NATRECOR (Nesiritide), ARANESP (Darbepoetin Alfa), NEULASTA (Pegfilgrastim), NEUMEGA (Oprelvekin), NEUPOGEN (Filgrastim), NORDITROPIN CARTRIDGES (Somatropin), NOVOSEVEN (Eptacog Alfa), NUTROPIN AQ (Somatropin), Oncaspar (Pegaspargase), ONTAK (Denileukin Difitox), ORTHOCLONEOKT (muromonab-CD3), OVIDREL (choriogonadotropin alfa), PEGASYS (peginterferon alfa-2a), PROLEUKIN (aldesleukin), PULMOZYME (dornase alfa), Retavase (reteplase), REBETOL® (ribavirin) and REBETRON combination therapy including INTRON® A (interferon alfa-2b), REBIF (interferon beta-1a), REFACTO (antihemophilic factor), REFLUDAN (lepirudin), REMICADE (infliximab), REOPRO (abciximab) ROFERON®-A (interferon alpha-2a), SIMULECT (basiliximab), SOMAVERT (pegvisomant), SYNAGIS® (palivizumab), Stemben (ansestim, stem cell factor), THYROGEN, INTRON® A (interferon alpha-2b), PEG-INTRON® (peginterferon alpha-2b), XIGRIS® (activated drotrecogin alfa), XOLAIR® (omalizumab), ZENAPAX® (daclizumab), and ZEVALIN® (ibritumomab tiuxetan).

[0045] 29. Any of the above or below compositions, wherein the protein is Ab-hCD22 or a fragment thereof, or a mutant, derivative, or variant of Ab-hCD22 or a fragment thereof; Ab-hIL4R or a fragment thereof, or a mutant, derivative, or variant of Ab-hIL4R or a fragment thereof; Ab-hOPGL or a fragment thereof, or a mutant, derivative, or variant of Ab-hOPGL or a fragment thereof; Ab-hB7RP1 or a fragment thereof, or a mutant, derivative, or variant of Ab-hB7RP1 or a fragment thereof.

[0046] 30. Any of the above or below compositions, wherein the protein is Ab-hCD22 or Ab-hIL4R or Ab-hOPGL or Ab-hB7RP1.

[0047] 31. Any of the above or below compositions comprising a protein and a solvent, wherein the protein has a buffering capacity per unit volume and per pH unit that is equal to or greater than the buffering capacity of a 4.0 mM aqueous sodium acetate solution over the pH range of 4.0 to 5.0 or pH 5.0 to 5.5, which buffering capacity is measured in particular by the method described in Examples 1 and 2, and further wherein the buffering capacity per unit volume and per pH unit of the composition excluding the protein is equal to or less than the buffering capacity of a 2.0 mM aqueous sodium acetate solution over the same pH range as above, preferably measured by the same method.

[0048] 32. Any of the above or below compositions comprising a protein and a solvent, wherein at the pH of the composition, the buffering capacity of the protein is at least 1.63 mEq / l for a pH change of the composition of ±1 pH unit, and the buffering capacity of the composition excluding the protein is equal to or less than 0.81 mEq / l for a pH change of ±1 pH unit at the pH of the composition.

[0049] 33. A lyophilisate which upon reconstitution gives any of the above or below compositions.

[0050] 34. A kit comprising, in one or more containers, any of the compositions or lyophilisates described above or below and instructions for their use.

[0051] 35. A method for producing any of the above or below described compositions or lyophilisates, comprising removing residual buffer using a counterion.

[0052] 36. A method for producing any of the above or below described compositions or lyophilisates, comprising removing residual buffer using any one or more of chromatography, dialysis, and / or cross-flow filtration in the presence of counterions.

[0053] 37. A method for producing any of the above or below described compositions or lyophilisates, comprising removing residual buffer using cross-flow filtration.

[0054] 38. A method for producing any of the above or below described compositions or lyophilisates, comprising a step of dialysis against a solution having a pH equal to or lower than the pH of the formulation, and, if necessary, thereafter adjusting the pH by addition of dilute acid or base.

[0055] 39. A method for treating a subject comprising administering to the subject any of the above or below compositions comprising a reconstituted lyophilisate in a therapeutically effective amount and by a route. [Brief description of the drawings]

[0056] [Figure 1] FIG. 1 depicts titration data and buffer capacity as a function of concentration for sodium acetate standard buffers over the pH range 5.0-4.0. Panel A is a graph depicting pH change upon acid titration of several different concentrations of standard sodium acetate buffers as described in Example 1. pH is shown on the vertical axis. The amount of acid added to each solution is shown on the horizontal axis in microequivalents of HCl added per ml of solution (μEq / ml). A linear least squares slope is shown for each data set. Acetate concentration is shown in the inset. Panel B is a graph depicting buffer capacity of acetate buffers over an acidic pH range as determined from the titration data depicted in Panel A as described in Example 1. Buffer capacity is shown on the vertical axis as microequivalents of acid / ml of buffer / unit change in pH (μEq / ml-pH). Acetate concentration is shown on the horizontal axis in mM. [Diagram 2]FIG. 2 depicts titration data and buffer capacity as a function of concentration for sodium acetate standard buffers spanning pH 5.0-5.5. Panel A is a graph depicting pH change upon base titration of several different concentrations of standard sodium acetate buffers as described in Example 2. pH is shown on the vertical axis. The amount of base added to each solution is shown on the horizontal axis in microequivalents of NaOH added per ml of solution (μEq / ml). A linear least squares slope line is plotted for each data set. Acetate concentration is shown in the inset. Panel B is a graph depicting buffer capacity of acetate buffers spanning a basic pH range, as determined from the titration data depicted in Panel A, and described in Example 2. Buffer capacity is shown on the vertical axis as microequivalents of base / ml of buffer / unit change in pH (μEq / ml-pH). Acetate concentration is shown on the horizontal axis in mM. [Diagram 3] 3 depicts the measurement of acetate concentration in acetate buffer standards as described in Example 3. The graph shows the standard curve for the measurement with peak area on the vertical axis and acetate concentration on the horizontal axis. The nominal and measured amounts of acetate in the solutions used for the empirical measurement of buffer capacity are tabulated below the graph. [Figure 4] FIG. 4 is a graph showing pH change upon acid titration of several different concentrations of Ab-hOPGL over the pH range of 5.0 to 4.0, as described in Example 4. pH is shown on the vertical axis. The amount of acid added to the solution is shown on the horizontal axis in microequivalents of HCl added per ml of buffer (μEq / ml). A linear least squares fit slope is shown for each data set. Ab-hOPGL concentrations are shown in the inset. [Diagram 5] FIG. 5 is a graph showing pH change upon base titration of several different concentrations of Ab-hOPGL over the pH range of 5.0-6.0, as described in Example 5. pH is shown on the vertical axis. The amount of base added to the solution is shown on the horizontal axis in microequivalents of NaOH added per ml of buffer solution (μEq / ml). A linear least squares fit slope line is plotted for each data set. Ab-hOPGL concentrations are shown in the inset. [Figure 6]6 shows the residual acetate levels in Ab-hOPGL solutions used to measure buffer capacity. The graph shows the standard curve used for acetate measurement as described in Example 6. The nominal and experimentally determined acetate concentrations in the solutions are tabulated below the graph. [Figure 7] FIG. 7 is a graph showing the buffer capacity of Ab-hOPGL ± residual acetate over the pH range 5.0-4.0. The data was obtained as described in Example 7. The top line shows the buffer capacity of Ab-hOPGL with residual acetate. The bottom line shows the buffer capacity of Ab-hOPGL adjusted for residual acetate. The vertical axis shows the buffer capacity in microequivalents of acid / ml of Ab-hOPGL solution / pH unit (μEq / ml-pH). The horizontal axis shows the concentration of Ab-hOPGL in mg / ml. The buffer capacities of standard acetate buffers of different concentrations described in Example 1 are represented as horizontal lines. The buffer concentrations are shown above the lines. [Figure 8] FIG. 8 is a graph showing the buffer capacity of Ab-hOPGL ± residual acetate in the basic pH range, pH 5.0-6.0. Data was obtained as described in Example 8. The top line shows the buffer capacity of Ab-hOPGL with residual acetate. The bottom line shows the buffer capacity of Ab-hOPGL adjusted for residual acetate. The vertical axis shows the buffer capacity in microequivalents of added base / ml of buffer / pH unit (μEq / ml-pH). The horizontal axis shows the concentration of Ab-hOPGL in mg / ml. The buffer capacities of several concentrations of standard sodium acetate buffers described in Example 2 are shown as horizontal lines. Acetate concentrations are indicated above each line. [Figure 9]FIG. 9 depicts a pair of graphs of pH and Ab-hOPGL stability in self-buffered and conventionally buffered formulations. Panel A depicts the stability of self-buffered Ab-hOPGL, Ab-hOPGL formulated in acetate, and Ab-hOPGL formulated in glutamate as a function of storage time at 4° C. for 6 months. The vertical axis depicts Ab-hOPGL stability as % Ab-hOPGL monomer as measured by SE-HPLC. Storage time is shown on the horizontal axis. Panel B depicts the pH of the same three formulations measured over the same time periods. Protein stability measurements and pH measurements are described in Example 9. [Figure 10] FIG. 10 presents titration curves and buffer capacities over the pH range 5.0-4.0 for several concentrations of self-buffered Ab-hB7RP formulations. Panel A presents the titration data. pH is shown on the vertical axis. The amount of acid added to the solution is shown on the horizontal axis in microequivalents of HCl added / ml of buffer (μEq / ml). A linear least squares fit slope is shown for each data set. The concentration of Ab-hB7RP1 is shown in the inset. Panel B presents the buffer capacity of Ab-hB7RP1 formulations. The top line shows the buffer capacity of the formulation including the contribution of residual acetate. The bottom line shows the buffer capacity of the formulation after subtracting the contribution of residual acetate based on the SE-HPLC measurements described in Example 3. A linear least squares fit slope is shown for two data sets. The vertical axis shows the buffer capacity in microequivalents of acid / ml of buffer / pH units (μEq / ml-pH). The concentration of Ab-hB7RP1 is shown on the horizontal axis in mg / ml. The buffering capacity of standard sodium acetate buffers at several concentrations described in Example 1 is shown as dashed horizontal lines. The acetate buffer concentrations are shown below each line. The results were obtained as described in Example 10. [Figure 11]FIG. 11 presents titration curves and buffering capacities over the pH range 5.0-6.0 for several concentrations of self-buffered Ab-hB7RP1 formulations. Panel A shows the titration data. pH is shown on the vertical axis. The amount of base added to the solution is shown on the horizontal axis in microequivalents of NaOH added / ml of buffer (μEq / ml). Linear least squares slopes are shown for each data set. Ab-hB7RP1 concentrations are shown in the inset. Panel B presents the buffering capacities of Ab-hB7RP1 formulations. The top line shows the buffering capacity of the above formulation including residual acetate. The bottom line shows the buffering capacity of the formulation adjusted to remove the contribution of residual acetate. Linear least squares slopes are shown for two data sets. The vertical axis shows the buffering capacity in microequivalents of base / ml of buffer solution / pH unit (μEq / ml-pH). The concentration of Ab-hB7RP1 is shown on the horizontal axis in mg / ml. The buffering capacity of standard sodium acetate buffers at several concentrations as described in Example 2 is shown by the dashed horizontal lines. The acetate buffer concentrations are indicated above each line. Results were obtained as described in Example 11. [Figure 12] FIG. 12 depicts the stability of Ab-hB7RP1 in self-buffered and conventionally buffered formulations at 4° C. and 29° C. Panel A depicts the stability of self-buffered Ab-hB7RP1, Ab-hB7RP1 formulated in acetate buffer, and Ab-hB7RP1 formulated in glutamate as a function of storage time at 4° C. for 6 months. The vertical axis represents the percentage of Ab-hB7RP1 monomer in the samples as measured by SE-HPLC. Time is indicated on the horizontal axis. Panel B depicts the stability of the same three formulations as above as a function of storage over the same time periods at 29° C. The vertical and horizontal axes of Panel B are the same as those of Panel A. Measurement of protein stability by HPLC-SE is described in Example 12. [Figure 13] 13 depicts the pH stability of self-buffered formulations of Ab-hB7RP1 at 4° C. and 29° C. The vertical axis represents pH. Time (weeks) is shown on the horizontal axis. The temperature of the data set is shown in the inset. The data was obtained as described in Example 13. [Figure 14]FIG. 14 depicts the buffering capacity of self-buffered formulations of Ab-hCD22 as a function of Ab-hCD22 concentration over the pH range 4.0-6.0. Panel A depicts the buffering capacity of self-buffered Ab-hCD22 formulations as a function of Ab-hCD22 concentration over the pH range 4.0-5.0. Panel B depicts the buffering capacity of self-buffered Ab-hCD22 formulations as a function of concentration over the pH range 5.0-6.0. In both panels, the vertical axis represents the buffering capacity in microequivalents of base / ml of buffer solution / pH unit (μEq / ml-pH) and the horizontal axis represents the Ab-hCD22 concentration in mg / ml. For reference, the buffering capacity of 10 mM sodium acetate as described in Example 1 is shown in both panels as a dashed horizontal line. The results shown in this figure were obtained as described in Example 14. [Figure 15] FIG. 15 depicts titration curves and buffering capacities for several concentrations of self-buffered Ab-hIL4R formulations over the pH range 5.0-4.0. Panel A depicts titration data. pH is shown on the vertical axis. The amount of acid added to the solution is shown on the horizontal axis in microequivalents of HCl added / ml of buffer solution (μEq / ml). A linear least squares fit slope is shown for each data set. Ab-hIL4R concentrations are shown in the inset. Panel B depicts buffering capacity of Ab-hIL4R as a function of concentration. A linear least squares fit slope is shown for each data set. The vertical axis depicts buffering capacity in microequivalents of base / ml of buffer solution / pH units (μEq / ml-pH). The concentration of Ab-hIL4R is shown on the horizontal axis in mg / ml. The buffering capacity of several concentrations of standard sodium acetate buffers described in Example 1 are shown as dotted horizontal lines. The acetate buffer concentrations are shown above each line. Results were obtained as described in Example 15. [Figure 16]FIG. 16 presents titration curves and buffering capacities for several concentrations of self-buffered Ab-hIL4R formulations over the pH 5.0-6.0 range. Panel A shows the titration data. pH is shown on the vertical axis. The amount of base added to the solution is shown on the horizontal axis in microequivalents of NaOH added / ml of buffer solution (μEq / ml). A linear least squares fit slope is shown for each data set. Ab-hIL4R concentrations are shown in the inset. Panel B presents the buffering capacity of Ab-hIL4R as a function of concentration. A linear least squares fit slope is shown for this data set. The vertical axis represents buffering capacity in microequivalents of base / ml of buffer solution / pH units (μEq / ml-pH). The concentration of Ab-hIL4R is shown on the horizontal axis in mg / ml. The buffering capacity of several concentrations of standard sodium acetate buffers described in Example 2 are shown as dashed horizontal lines. The acetate buffer concentrations are shown above each line. Results were obtained as described in Example 16. [Figure 17] FIG. 17 depicts the stability of Ab-hIL4R and pH as a function of time in acetate buffer and self-buffered formulations of Ab-hIL4R at 37° C. Panel A is a bar graph showing the stability of Ab-hIL4R over 4 weeks at 37° C. The vertical axis shows stability as percent monomeric Ab-hIL4R as measured by SE-HPLC. The horizontal axis shows storage time in weeks. The inset identifies the acetate data and the self-buffered formulation data. Panel B shows the pH stability of the same formulations as above, for the same conditions and time periods. pH is shown on the vertical axis. Storage time (weeks) is shown on the horizontal axis. Data for acetate and self-buffered formulations are shown in the inset. This data was obtained as described in Example 17. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] Glossary The meanings ascribed to various terms and phrases used in this specification are specifically explained below.

[0058] As used herein, the term "a" or "an" means "at least one;" "one or more."

[0059] The term "about" means ±20% unless otherwise specified. For example, in this specification, about 100 means 80 to 120, about 5 means 4 to 6, about 0.3 means 0.24 to 0.36, and about 60% means 48% to 72% (not 40% to 80%).

[0060] As used herein, the term "agonist" refers to a molecular entity that is distinct from the corresponding stimulatory ligand, but has the same stimulatory effect. For example, with respect to a hormone that stimulates activity by binding to the corresponding hormone receptor, an agonist is a chemically distinct entity that binds to the hormone receptor and stimulates its activity (although an agonist acts through another mechanism).

[0061] As used herein, the term "antagonist" refers to a molecular entity that has a different and opposite action from the corresponding ligand. For example, one type of antagonist of a hormone that stimulates activity by binding to the corresponding hormone receptor (although antagonists function through other mechanisms) is a chemical entity that, unlike the hormone, binds to the hormone receptor but does not stimulate the activity caused by hormone binding, thereby inhibiting the effector activity of the hormone.

[0062] The term "antibody" is used herein in accordance with its conventional meaning in the art of biochemistry and biotechnology.

[0063] Antibodies within the meaning of the term as used herein are antibodies isolated from biological sources, including monoclonal and polyclonal antibodies, antibodies produced by recombinant DNA technology (also referred to herein as recombinant antibodies), such as antibodies produced by processes involving the activation of endogenous genes and antibodies produced by processes involving the expression of exogenous expression constructs (e.g., antibodies produced in cell culture, and antibodies produced in transgenic plants and animals, and antibodies produced by processes involving chemical synthesis, such as peptide synthesis and semi-synthesis).Specifically chimeric and hybrid antibodies are also included within the scope of the term as used herein, unless expressly indicated otherwise.

[0064] The prototypical antibody is a tetrameric glycoprotein consisting of two identical light-heavy chain dimers linked by disulfide bonds. In vertebrates, there are two types of light chains: kappa and lambda. Each light chain consists of a constant region and a variable region. The two light chains are distinguished by the sequence of the constant region. In vertebrates, there are five types of heavy chains: alpha, delta, epsilon, gamma, and mu. Each heavy chain consists of a variable region and three constant regions. The five types of heavy chains define the five types (isotypes) of vertebrate antibodies: IgA, IgD, IgE, IgG, and IgM. Each isotype is composed of (a) two alpha, delta, epsilon, gamma, or mu heavy chains, and (b) two kappa or two lambda light chains. Each type of heavy chain binds both types of light chains, but the two light chains in a given molecule are either both kappa or both lambda. IgD, IgE, and IgG usually occur as "free" heterotetrameric glycoproteins. IgA and IgM usually occur as complexes containing several IgA or several IgM heterotetramers associated with a "J" chain polypeptide. Some vertebrate isotypes are distinguished from each other and divided into subclasses by differences in the constant region sequences. There are four human IgG subclasses, e.g., IgG1, IgG2, IgG3, and IgG4, and two IgA subclasses, IgA1 and IgA2. All these antibodies, as well as other antibodies not specifically mentioned above, are included within the meaning of the term "antibody" as used herein.

[0065] The term "antibody" further includes amino acid sequence variants of any of the above, as further described elsewhere herein.

[0066] As used herein, "antibody-derived" refers to any protein made from an antibody and any protein designed based on an antibody. The term includes within its meaning proteins made using all or a portion of an antibody, proteins that contain all or a portion of an antibody, and proteins that are designed in whole or in part based on all or a portion of an antibody. "Antibody-derived" proteins include, but are not limited to, Fc, Fab, and Fab. 2 Fragments and proteins containing them, V H Domain Fragment and V L These include domain fragments and proteins comprising these, other proteins comprising the variable and / or constant regions of antibodies in whole or in part, scFv intrabodies, maxibodies, minibodies, diabodies, amino acid sequence variants of the above, as well as a variety of other such molecules, including, but not limited to, other proteins described elsewhere herein.

[0067] "Antibody-related" as used herein means any protein or mimetic whose structure, function, or design resembles an antibody or any portion of an antibody. An "antibody-related" protein as the term is used herein is an "antibody-derived" protein as described above. It should be noted that the terms "antibody-derived" and "antibody-related" overlap substantially. Both terms apply to many such proteins. Examples of "antibody-related" proteins, without intending to be limiting in this respect, are peptide antibodies and receptorbodies. Other examples of "antibody-related" proteins are described elsewhere herein.

[0068] As used herein, unless otherwise specified, "antibody polypeptide" refers to a polypeptide that is a part of an antibody (e.g., a light chain polypeptide, a heavy chain polypeptide, and a J chain polypeptide), including, among other examples, fragments, derivatives, and variants thereof, and related polypeptides.

[0069] The term "approximately" has the same meaning as "about" unless otherwise specified.

[0070] "Binding moiety" means a portion of a molecule or molecular complex that specifically binds to a portion of another molecule or molecular complex. The binding moiety may be the same as or different from the portion of the molecule or molecular complex to which it binds. The binding moiety may be the entire molecule or molecular complex.

[0071] "Specifically binds" is used herein according to its ordinary meaning in the art, and unless otherwise specified, means that the binding to a particular specific moiety is stronger than the binding to other general moieties, is stronger than the non-specific binding that may occur to a wide range of moieties, and that the binding is selective for the particular moiety and does not bind to a similar extent to other moieties. In the extreme case of specific binding, very strong binding occurs with a single type of moiety and no non-specific binding occurs with any other moieties.

[0072] The term "co-administer" refers to the administration of two or more agents in combination with one another, including simultaneous and / or sequential administration.

[0073] As used herein, "cognate" means complementary, fitting together, or matching, such as two pieces of a jigsaw puzzle that fit together, the cylinder mechanism of a lock and the key that opens it, the substrate binding site of an enzyme and the substrate for that enzyme, and a target and a target binding protein that specifically binds to it.

[0074] By "cognate binding moiety" herein is meant binding moieties that specifically bind to each other. Typically, but not always, it means a pair of binding moieties that specifically bind to each other. Moieties that participate in highly selective binding between specific ligands and ligand receptors provide specific examples of cognate binding moieties. Another example is provided by moieties that bind to antigens and antibodies.

[0075] The term "composition" refers to any composition, such as a formulation, that contains one or more components.

[0076] "Consisting of" is synonymous with "including" (see below).

[0077] "Comprising" means containing without further reservation, limitation, or exclusion as to anything else that may or may not be included. For example, "a composition comprising x and y" means any composition that contains x and y, regardless of what else that composition may contain. Similarly, "a process comprising x" is any process in which x is performed, regardless of what else may occur.

[0078] The term "concentration" is used herein according to its well-known meaning in the art, and refers to the amount of an item in a given amount of a mixture containing that item, typically expressed as a percentage.For example, the concentration of a solute (such as a protein in a solution) can be expressed in many ways, such as (but not limited to): (A) weight percent (i) = weight of solute per 100 parts of solvent volume; (B) weight percent (ii) = weight of solute per 100 parts of total weight; (C) weight percent (iii) = weight of solute per 100 parts of solvent weight; (D) mass percent = mass of solute per 100 parts of solution weight; (E) molar ratio = moles of solute per total moles of all components; (F) molar concentration = moles of solute per liter of solution (i.e., solvent to which solute is added); (G) molality = moles of solute per Kg of solvent; and (H) molality = moles of solute per liter of solvent.

[0079] As used herein, the term "control region" is used according to its well-known meaning in the art, and unless otherwise specified, refers to a region in DNA or protein that is involved in controlling one or more functions or activities. For example, an "expression control region" in relation to the control of gene expression means a region in DNA that is required for proper transcription to occur, is involved in regulation when transcription occurs, and is involved in how efficiently transcription is stopped when transcription is stopped.

[0080] As used herein, "de novo" is used according to its well-known meaning in the art and means something newly created. For example, a de novo amino acid sequence is a sequence that is not derived from a natural amino acid sequence, but such a de novo sequence may have similarity to a natural sequence. De novo amino acid sequences can be generated, for example, by predictive design, combinatorial methods, and selection methods. These amino acids can be produced, for example, by chemical synthesis, semi-synthesis, and various recombinant DNA techniques, all of which are well known to those skilled in the art.

[0081] The term "deleterious" as used herein means "harmful." Examples include deleterious effects, including, for example, the harmful effects of disease progression and harmful side effects of treatment.

[0082] As used herein, "derivative" refers to something derived in substance, form, or design, e.g., a polypeptide that is based on a reference polypeptide but differs from the reference polypeptide by alterations in the amino acid sequence, fusion with another polypeptide, or covalent modifications.

[0083] A "disease" is a condition or symptom that adversely affects the health of a subject.

[0084] "Disorder" is a disorder or condition that impairs health.

[0085] As used herein, the term "dysfunction" refers to a disturbance, disease, or deleterious effect of a normally normal process.

[0086] "Effective amount" usually means an amount that produces a desired local or systemic effect. For example, an effective amount is an amount that is sufficient to produce a beneficial or desired clinical result. This effective amount can be provided all at once in a single administration, or in a divided amount that gives an effective amount in several administrations. The exact determination of the amount that is considered effective can be based on each subject's individual factors, such as the subject's size, age, injury, and / or disease or injury being treated, and the amount of time since the injury occurred or the disease developed. A person skilled in the art will be able to determine the effective amount for a given subject based on these considerations that are routine in the art. As used herein, "effective dosage" means the same as "effective amount."

[0087] "Effective route" generally refers to a route that provides for delivery of an agent to a desired compartment, system, or location. For example, an effective route is one through which an agent can be administered to provide a sufficient amount of agent at a desired site of action to achieve a beneficial or desired clinical result.

[0088] As used herein, "endogenous" (e.g., endogenous gene) is used to refer to, e.g., genes and other aspects of DNA, e.g., genomes and control regions that occur naturally in an organism, unless otherwise indicated.

[0089] As used herein, unless otherwise indicated, "exogenous" (e.g., exogenous gene) is typically used to mean DNA that originates from an outside source, e.g., DNA that is introduced into a cell and integrated into its genome.

[0090] "FBS" means fetal bovine serum.

[0091] A "formulation" means a combination of at least one active ingredient with one or more other ingredients for one or more specific uses, such as storage, further processing, sale, and / or administration to a subject (e.g., administration of a particular agent in a particular amount to a subject by a particular route to treat a particular disease).

[0092] As used herein, a "fragment" refers to a portion of a larger entity, such as a portion of a protein (e.g., a polypeptide consisting of less than the complete amino acid sequence of a larger polypeptide). As used herein, the term includes fragments formed by terminal deletions and fragments formed by internal deletions (e.g., fragments in which two or more non-contiguous portions of a polypeptide are joined internally to form a smaller polypeptide that is a fragment of the original protein).

[0093] As used herein, a "fusion protein" refers to a protein formed by fusing all or part of two polypeptides, which may be the same or different. A typical fusion protein is produced by end-to-end joining of nucleotides encoding the two (or more) polypeptides by recombinant DNA techniques.

[0094] As used herein, the term "genetically engineered" means produced using artificial methods of genetic modification, such as recombinant DNA techniques, classical methods of genetic engineering, chemical methods, a combination of all three, or other methods.

[0095] As used herein, the term "homolog" means having homology to another entity, for example a protein that is homologous to another protein. "Homologous" means similar in structure or function.

[0096] As used herein, "ionization" refers to a change in at least one charge in the net charge on a substance, including the loss or gain of charge, e.g., from HOAc to OAc. - and H +Examples include the ionization of acetic acid in low pH solutions to

[0097] As used herein, "k" denotes an equilibrium coefficient according to its standard meaning in the chemical arts.

[0098] In this specification, "k a " denotes the dissociation constant of a particular hydrogen of a molecule, e.g., the dissociation constant of the acidic hydrogen of acetic acid, in accordance with the standard meaning in the chemical arts.

[0099] In this specification, "k d " denotes the dissociation constant of a pair of chemical entities (or moieties), according to the standard meaning in the chemical arts.

[0100] "Kit" means a collection of items used together for a given purpose or purposes.

[0101] As used herein, "ligand" refers to a molecular entity that selectively and stoichiometrically binds to one or more specific sites on one or more other molecular entities. The binding is typically non-covalent, but may be covalent. A few examples, among many others, are: (a) antigens, which typically bind non-covalently to binding sites on cognate antibodies; (b) hormones, which typically bind non-covalently to hormone receptors; (c) lectins, which non-covalently bind to specific sugars; (d) biotin, which typically binds non-covalently to multiple sites on avidin and other avidin-like proteins; (e) hormone antagonists, which bind to hormone receptors and inhibit their activity and / or the activity of the corresponding hormone; and (f) hormone agonists, which also bind to hormone receptors but promote these activities.

[0102] By "ligand binding moiety" herein is meant a molecular entity that binds to a ligand, typically a part of, or a molecular entity derived from, a larger molecular entity that binds to the ligand.

[0103] As used herein, "ligand binding protein" refers to a protein that binds to a ligand.

[0104] As used herein, a "ligand moiety" refers to a molecular entity that binds to a ligand-binding molecular entity in much the same manner as a corresponding ligand. The ligand moiety may be the whole or a portion of a ligand, a moiety derived from a ligand, or a moiety created de novo. Typically, however, the ligand moiety is in a mode that binds more or less exclusively to a corresponding ligand-binding entity. A ligand moiety need not include, and the term does not normally imply, structural features other than those required for ligand binding.

[0105] As used herein, "mEq" means milliequivalents.

[0106] As used herein, "μEq" means microequivalent.

[0107] As used herein, "mimetic" refers to a chemical entity that has a structural or functional feature of another, usually unrelated, chemical entity. For example, certain hormone mimetics are non-peptide organic molecules that bind to corresponding receptors in a manner similar to that of the corresponding hormones.

[0108] "mM" is millimolar; 10 -3 It means moles / l.

[0109] As used herein, a "modified protein," "modified polypeptide," or "modified fragment" refers to a protein or polypeptide, or a fragment of a protein or polypeptide, that contains chemical moieties (structures) other than those of the 20 naturally occurring amino acids that form naturally occurring proteins. Modifications are most often covalently attached, but can also be non-covalently attached to proteins or other polypeptides, e.g., fragments of proteins.

[0110] As used herein, the term "portion" refers to a molecular entity that embodies a particular structure and / or function without exogenous components. For example, often only a small portion of a ligand-binding protein is involved in ligand binding. This portion of the protein, whether encoded contiguously or discontinuously, is an example of a ligand-binding portion.

[0111] "Natural" means occurring in nature without human intervention.

[0112] "Non-naturally occurring" means not occurring in nature, or, if occurring in nature, not in the natural state, environment, situation, etc.

[0113] "PBS" means phosphate buffered saline.

[0114] "Peptide antibodies" are antibodies with C H 1. C L , V H , and V L The Fc domain of an antibody excluding the C domain and Fab and F(ab)2 (i.e., H 2 and C H Peptide antibodies refer to molecules comprising one or more peptides, preferably pharmacologically active peptides, particularly preferably randomly generated pharmacologically active peptides, in which the Fc domain is linked to one or more peptides, preferably pharmacologically active peptides, particularly preferably randomly generated pharmacologically active peptides. The production of peptide antibodies is generally described in PCT Publication WO00 / 24782, published May 4, 2000, which is incorporated by reference in its entirety, particularly with respect to the structure, synthesis, properties and uses of peptide antibodies.

[0115] As used herein, "peptide" is synonymous with polypeptide; frequently, but not necessarily, it is used in reference to relatively short polypeptides.

[0116] "pH" is as follows: pH = -log[H 3 O+] It is used according to the well-known and commonly used definition.

[0117] As used herein, the term "pharmaceutical" means acceptable for use in humans or non-human subjects for treatment, particularly for use in humans, and approved for use in humans or non-human subjects for treatment by a regulatory agency legally empowered to regulate its use, such as the U.S. Food and Drug Administration, the European Medicines Agency, the Ministry of Health, Labor and Welfare of Japan, or other regulatory agencies listed in R. Ng, DRUGS: FROM DISCOVERY TO APPROVAL, Wiley-Liss (Hoboken, NJ) (2004). The disclosures of the above documents are incorporated herein by reference in their entirety, particularly with respect to the regulatory agencies involved in drug approval as listed in Chapter 7. As used herein, the phrase "the composition is approved for pharmaceutical use by an authority legally empowered to grant approval for pharmaceutical use" refers to a body or agency, etc. established by law that is charged with the responsibility and power to regulate and approve the use of drugs in humans and, optionally, non-humans. Approval by any one such agency satisfies this requirement. The approval body need not, for example, be the approval body of the country in violation. Examples of such bodies include the U.S. Food and Drug Administration and other agencies described herein above.

[0118] The term "pharmaceutical product" as used herein may also refer to products manufactured in accordance with good manufacturing practices as set forth in Chapters 9 and 10 of R. Ng, DRUGS: FROM DISCOVERY TO APPROVAL, Wiley-Liss (Hoboken, NJ) (2004), the disclosures of which are incorporated herein by reference in their entirety, particularly in the portions relating to good manufacturing practices for pharmaceutical protein formulations as set forth in Chapters 9 and 10.

[0119] As used herein, the term "pharmaceutical acceptable" is used in accordance with its well-known meaning in the art to mean acceptable for medical or veterinary use, preferably for human medical use, and particularly as used in the context of "pharmaceutical" as approved for such use by the U.S. Food and Drug Administration or other agencies mentioned above.

[0120] A "polypeptide" is considered a "protein."

[0121] As used herein, the term "precursor" is used according to its well-known meaning in the art to refer to an entity from which another entity is derived. For example, a precursor protein is a protein that undergoes processing, such as proteolytic cleavage or modification, to give rise to another precursor protein (which undergoes further processing) or to a mature protein.

[0122] As used herein, "protein" refers to a polypeptide or complex of polypeptides according to its well-known meaning in the art. As used herein, "protein" includes both linear and branched polypeptides. "Protein" includes unmodified polypeptides and modified polypeptides containing natural and non-naturally occurring modifications. Such modifications include chemical modifications of the termini, peptide backbones and amino acid side chains; amino acid substitutions, deletions and additions; and introduction of unusual amino acids and other moieties, to name a few. The protein also includes "engineered" polypeptides and complexes thereof, including, but not limited to, any polypeptide or complex of polypeptides whose structure has been artificially altered, for example, by recombinant DNA technology, chemical synthesis, and / or covalent modification (e.g., artificial alteration of the amino acid sequence and / or post-translational modification).

[0123] "Protonated" means the addition of at least one hydrogen.

[0124] "Self-buffering" refers to the ability of a substance, such as a pharmaceutical protein, in the absence of other buffering agents, to withstand pH changes sufficient for a given application.

[0125] As used herein, "semi-de novo" means (a) partially designed according to specific criteria and / or produced from precursors, and (b) partially designed without reference to specific criteria (e.g., designed solely by general principles and without any specific criteria). For example, a polypeptide may be made by producing a first peptide in a microbial expression system, producing a second peptide by chemical synthesis, and then combining the two peptides to form the polypeptide.

[0126] As used herein, "semi-synthetic" refers to a combination of chemical and non-chemical synthetic methods.

[0127] "Subject" means a vertebrate, such as a mammal (e.g., a human). Mammals include, but are not limited to, humans, farm animals, sport animals, and pets. Subjects in need of treatment with the methods and / or compositions of the invention include subjects suffering from a disorder, dysfunction, or disease or side effects thereof, or side effects of treatment thereof.

[0128] "Substantially" is used herein according to its plain and / or ordinary definition to mean "a large degree" or "a large degree." For example, "substantially complete" means "complete to a large degree" or "complete to a large degree." As a further example, "substantially free of residue" means "free of residue to a large degree" or "free of residue to a large degree." When numerical precision is required, depending on the context, the term "substantially" as used herein means at least 80% or more, particularly 90% or more, and very particularly 95% or more.

[0129] "Therapeutically effective" is used herein in accordance with its well-known meaning in the art to mean achieving an improvement in a subject's prognosis or condition or otherwise achieving a therapeutic objective, e.g., a reduction in the rate of disease progression despite the subject's condition continuing to worsen.

[0130] "Therapeutically effective amount" is generally used to qualify the amount of drug to include the amount that achieves the improvement of serious disorders. For example, an effective tumor treatment agent will prolong the survival rate of a subject, inhibit the rapid cell proliferation associated with a tumor, or affect tumor regression. A therapeutically effective treatment within the meaning of the term as used herein includes a treatment that improves the quality of life of a subject, even if it does not improve the outcome of the disease itself.

[0131] "Treate," "treating," or "treatment" are used broadly in the context of the present invention, and each of these terms specifically encompasses the prevention, amelioration, inhibition, or cure of a deficiency, dysfunction, disease, or other deleterious process (including processes that interfere with and / or result from the treatment).

[0132] As used herein, "variant" refers to, for example, a naturally occurring or synthetic form of a protein that differs structurally from the original protein, but is structurally and / or functionally related, e.g., an allelic variant, paralog, or homolog of the protein.

[0133] Detailed Description of the Invention The present invention provides for the first time particularly self-buffering protein formulations, particularly biopharmaceutical protein formulations, methods for producing the formulations, and particularly methods for using the formulations. Any protein that provides sufficient buffering capacity within the required pH range at a concentration suitable for its intended use can be produced as a self-buffering protein formulation of the invention. The present invention can be practiced with a variety of proteins, including both naturally occurring proteins and "engineered" proteins, particularly biopharmaceutical proteins, which are discussed further below.

[0134] The utility of proteins, particularly biopharmaceutical proteins, formulated as self-buffering compositions, particularly pharma- ceutical compositions, was not recognized prior to the inventions disclosed herein. The effect of proteins in regulating physiological pH has been recognized and has been studied. However, it has not been recognized until now that proteins, particularly biopharmaceutical proteins, can have sufficient buffering capacity to maintain the formulation within a desired pH range without the use of additional buffering agents.

[0135] Biopharmaceutical proteins for use in the United States are formulated as buffered solutions, unbuffered solutions, amorphous or crystalline suspensions, and lyophilizates.

[0136] Most buffered solution formulations use conventional buffering agents. Two proteins, Pulmozyme® and Humulin®, are formulated as solutions without conventional buffering agents. Neither of these proteins provide substantial self-buffering capacity in the formulations.

[0137] Pulmozyme® has a molecular weight of about 37,000 daltons and contains 5 histidines, 22 aspartic acids, and 12 glutamic acids among its 260 amino acids. The buffering capacity of this protein within 0.5 pH units at pH 6.3 is essentially determined by its histidine content. Based on this fact, the upper limit of the self-buffering capacity of the formulation is determined by the effective concentration of histidine residues, 0.15 mM. The molar concentration of aspartic acid and glutamic acid in the formulation is 0.9 mM. Thus, the total molar concentration of all three amino acids combined is only slightly more than 1 mM at the concentration of the formulation.

[0138] Humulin® is formulated at a concentration of 3.5 g / ml. The protein has a molecular weight of approximately 6,000 daltons and contains two aspartic acids, eight glutamic acids, and two histidines. None of these amino acids are particularly effective buffers at the pH of the formulation, 7.0-7.8.

[0139] At this concentration, pK a The molarity of histidine closest to the pH of the formulation is 1.16 mM.

[0140] The biopharmaceutical lyophilizates of the present invention are reconstituted before use to form a solution or suspension. Most lyophilizates contain conventional buffers that maintain the correct pH of the reconstituted formulation. Some other biopharmaceutical lyophilizates, which have low protein concentrations or require low (less than 3) or high (above 9.5) pH, are not effectively buffered.

[0141] Therefore, in current biopharmaceutical proteins, buffering is achieved using traditional buffering agents. The ability of proteins to buffer pharmaceutical protein formulations by themselves is not fully understood and has not been used in the manufacture of protein pharmaceuticals.

[0142] The measurement of the buffering capacity of a protein is primarily important for developing self-buffering protein formulations according to the present invention. In this regard, methods for measuring buffering capacity and methods for measuring buffering capacity of a protein are described below. To be able to provide comparability of the data, the protein buffering capacity must be expressed in comparable units and / or in relation to a buffer standard. The following section therefore describes the measurement criteria of pH and standards that meet these requirements according to the present invention.

[0143] 1. buffer The widely accepted definition of "buffering" is the resistance of a composition to a change in pH upon the addition of acid or base. Thus, buffering capacity is often defined as the ability of a composition to resist a change in pH.

[0144] Typically, buffer capacity is expressed in terms of the amount of strong acid or strong base required to change the pH of a composition by a given amount. Van Slyke provided the most widely used quantitative measure of buffer capacity. According to this measure, the buffer capacity of a solution is expressed as the amount of strong acid or strong base required to change the pH of 1 liter of said solution by 1 pH unit under standard temperature and pressure conditions.

[0145] According to this standard, for example, the buffer capacity of 1 liter of 5 mM acetic acid salt (HOAc) or 5 mM sodium acetate (NaOAc) dissolved in pure water at pH 4.76 is 4.09 x 10 -3 moles of strong monovalent base (i.e., 4.09 x 10 -3 equivalent of base) and can be calculated as follows:

[0146] The Henderson-Hasselbalch equation for the above solution is: pH = log {[5mM]NaOAc / [5mM]HOAc} + 4.76 Therefore, the concentration X of a strong monovalent base needed to raise the pH of this buffer is: 4.76~5.76 is 5.76 = log {[5 mM + X mM] NaOAc / [5 mM - X mM] HOAc} + 4.76, Therefore: 1.00 = log {[5mM + XmM] NaOAc / [5mM - XmM] HOAc} 10.0 = [5mM + XmM] NaOAc / [5mM - XmM] HOAc 10.0 = (5mM + XmM) / (5mM - XmM) 50 mM - 10X mM = 5 mM + X mM 11X mM = 45 mM X = 4.09 mM, and Per litre: (4.09 x 10 -3 moles / liter (1 liter) (1 equivalent / mol) = 4.09 x 10 -3 Generates an equivalent amount.

[0147] Therefore, by this standard, the buffering capacity of 1 liter of 10 mM acetate buffer containing 5 mM NaOAc and 5 mM HOAC in pure water at pH 4.76 is 4.09 x 10 base. -3 Conversely, the buffer capacity of the above solution is 4.09 milliequivalents of base / liter / pH unit, 4.09 microequivalents of base / milliliter / pH unit, 0.409 microequivalents of base / 100 microliters / pH unit, 40.9 nanomoles of base / 10 microliters / pH unit, and 4.09 nanomoles of base / microliter / pH unit.

[0148] The same calculations yield the following buffer capacities for other concentrations of this acetate buffer at pH 4.76: At 2 mM, the acetate buffer has a buffer capacity of 0.818 mEq / liter / pH unit. At 4 mM, the buffer capacity is 1.636 mEq / liter / pH unit. At 5 mM, the buffer capacity is 2.045 mEq / liter / pH unit. At 7.5 mM, the buffer capacity is 3.068 mEq / liter / pH unit. At 10 mM, the acetate buffer has a buffer capacity of 4.091 mEq / liter / pH unit. At 15 mM, the buffer capacity is 6.136 mEq / liter / pH unit.

[0149] Dissociation constant of acetic acid (pK a ) (pH 4.76), the acetate buffer solution has equimolar acetic acid and acetate (i.e., the pK a It is worth noting that, at 100°C, the acid and base are present in equal amounts. As a result, the pK of acetic acid is a The resistance of an acetate buffer to a change in pH (buffering capacity) at 20°C is the same for both the addition of an acid and a base. The acid-base equilibrium is determined by the pK a This is a general characteristic at a pH equal to

[0150] At any other pH, a buffer will contain different amounts of acid and base forms, so that the buffer's resistance to change upon addition of acid (i.e., its buffering capacity) will not be the same as its resistance to change upon addition of base. As a result, it is preferred to specify the capacity of such a buffer in terms of (i) the amount of acid required to lower the pH by one unit, and (ii) the amount of base required to raise the pH by one unit.

[0151] The separation of acids and bases in a buffer occurs for a given composition, e.g., a pH standard, and can be calculated at any pH and buffer concentration using the method described above in describing the buffer capacity of 10 mM NaOAc (containing equimolar amounts of acetic acid and sodium acetate) at pH 4.76 plus or minus. The results can be used to define the buffer capacity of a reference standard.

[0152] Thus, for example, the dissociation of acetic acid in a solution at pH 5.0 into acetic acid and acetate salts can be easily calculated using the above procedure, and from this value the buffer capacity can be calculated for both base and acid addition. Calculated in this way, the theoretical buffer capacity of a 10 mM sodium acetate buffer over pH 5.0-5.5 is about 2.1 mM / 0.5 pH unit, or about 4.2 mM / pH unit. In other words, the buffer capacity of the buffer is theoretically about 4.2 μEq / ml of buffer solution / pH unit change. Similarly, the theoretical buffer capacity of a 10 mM sodium acetate buffer over pH 5.0-4.0 is 4.9 mM, or in other words, 4.9 μEq / ml of buffer solution / pH unit change over the given pH range.

[0153] While such calculations are often quite useful, empirical standards and empirical measurements are preferred. Particularly preferred empirical standards are the sodium acetate buffers over the pH ranges 5.0-4.0 and 5.0-5.5, as exemplified in Examples 1 and 2. Particularly preferred are sodium acetate buffers according to Examples 1 and 2, which have a total acetate concentration of 10 mM, preferably 5 mM, and especially 4 mM, among other concentrations as set forth elsewhere herein.

[0154] As described above, an acetate buffer at pH 5.0 is more resistant to pH change upon addition of acid than to pH change upon addition of base. In a preferred empirical standard of buffer capacity, the buffer capacity of a standard acetate buffer is defined, for example, as follows: (i) the slope of the least squares regression line calculated for the base titration data of the buffer from pH 5.0 to pH 5.5, and (ii) the slope of the least squares regression line calculated for the acid titration data of the buffer from pH 5.0 to pH 4.0. Preparation of standard acetate buffers and measurement of their buffer capacity are described in Examples 1, 2 and 3. It will be understood that buffer capacity standards can be established and used using other suitable buffers using substantially the same method.

[0155] In measuring the buffering capacity of a self-buffering protein composition according to this invention, it is convenient to express this buffering capacity in terms of the concentration of a standard buffer having the same buffering capacity at the same pH. a In the case where a standard not listed above is used (e.g., a sodium acetate buffer initially at pH 5.0), a self-buffering composition of the present invention is defined as having a buffer capacity equal to or greater than that of the standard if either (or both) its buffer capacity upon base titration or its buffer capacity upon acid titration is equal to or greater than the corresponding buffer capacity of the standard.

[0156] The pH of a self-buffering protein composition according to the invention is typically determined by the pK aIt will be further appreciated that proteins are not located at the pK a and this contributes to the buffering capacity of the protein in a given pH range. Thus, the buffering capacity of the self-buffering protein formulation according to the present invention is preferably empirically measured by both acid titration and base titration over a given pH range change from the desired pH of the composition. In a preferred embodiment in this regard, the buffering capacity is measured by titrating with an acid and separately with a base over a change of ±1 pH unit from the starting pH of the formulation. In a particularly preferred embodiment, the titration data is collected for a pH change of ±0.5 pH unit. As described in the examples, the buffering capacity is the slope of the least squares regression line for the data of pH as a function of the equilibrium of the acid or base added to the composition over the titration range.

[0157] a. Empirical criteria and standards for buffer capacity In certain preferred embodiments of the invention, the measure of buffer capacity is an empirical standard. One preferred empirical standard in this regard is a specific volume of an aqueous solution containing a specific buffer at a specific concentration and either no components other than water or one or more other specific components, each at a specific concentration, at a specific temperature and a specific pH.

[0158] A particular standard that is particularly preferred for measuring buffer capacity according to various aspects and preferred embodiments of the present invention is pure water containing 10 mM sodium acetate, free of other components, at equilibrium with atmospheric pressure at 1 atm, pH 5.00, preferably expressed in equivalents / unit volume / pH units (e.g., μEq / ml-pH), as described in Examples 1 and 2. The buffer capacity of the standard must be empirically determined, as described in Examples 1, 2, and 3, and as further discussed elsewhere herein.

[0159] A particularly preferred specific standard for measuring buffer capacity according to various aspects and preferred embodiments of the present invention is pure water containing 10 mM sodium acetate, free of other components, at equilibrium with 1 atm atmosphere at 21° C., pH 4.76, as described in Examples 1 and 2, and preferably expressed as equivalents per unit volume per pH unit (e.g., μEq / ml-pH). The buffer capacity of the standard must be empirically determined as described in Examples 1, 2 and 3, and as discussed elsewhere herein. As described above, according to the Henderson-Hasselbalch equation, the calculated buffer capacity of this standard over the range of pH 4.76±1 pH unit is 4.09 microequivalents per milliliter per pH unit (4.09 μEq / ml-pH).

[0160] In accordance with various aspects and preferred embodiments of the invention in this regard, a variety of other buffers are available for use as standards in other pH ranges. Reference buffers, such as well-known buffers routinely used in analytical chemistry measurements, are particularly preferred in this regard. A variety of such buffers are presented in analytical chemistry textbooks and monographs on the accurate measurement of pH and buffer capacity.

[0161] Also useful in this regard are biological buffers, such as those described in, among other textbooks: TEITZ TEXTBOOK OF CLINICAL CHEMISTRY, 3rd ed., Burtis and Ashwood, eds., WB Saunders Company, Philadelphia, PA (1999), in particular Tables 50-13 to 50-16, the contents of which are incorporated herein by reference in their entirety with respect to buffering agents and buffer solutions according to the invention and their use as pH and / or buffer capacity standards; THE TOOLS OF BIOCHEMISTRY, Terrance G. Cooper, John Wiley & Sons, New York, NY (1977), in particular Chapter 1, pages 1-35, the contents of which are incorporated herein by reference in their entirety with respect to buffering agents and buffer solutions according to the invention and their use as pH and / or buffer capacity standards, very particularly with respect to Tables 1-3, 1-4, and 1-5 and the text related thereto; and PROTEIN PURIFICATION PRINCIPLES AND PRACTICE, 3rd ed., Robert K. Scopes, Springer-Verlag, New York, NY (1994), in particular pages 160-164, especially Tables 6.4 and 6.5 therein and the associated text, Chapter 12, Section 3, pages 324-333, especially Tables 12-4 and 12-5 therein and the associated text, and the entirety of Appendix C: Buffers for Use in Protein Chemistry, the contents of which are incorporated herein by reference in their entirety with respect to buffering agents and buffer solutions and their uses according to the present invention.

[0162] However, some of the gas dissolved in water is OH - and / or H 3 O +Since the buffer capacity of the standard solution of the present invention reacts with the atmosphere, the empirically measured buffer capacity of the standard solution of the present invention may vary somewhat from the theoretical value. Therefore, the definition of the standard of the present invention requires that the standard solution is in equilibrium with the atmosphere at 1 atmosphere pressure. Furthermore, the buffer standard must be kept in and in contact only with a material that does not change its composition or buffer capacity (e.g., a material that leaches acids, bases, or other reactants that can change the effective concentration or activity of the acetate buffer in any way that can change the buffer capacity). Considering both the atmospheric equilibrium and the inertness of the container as described above, the buffer capacity of the standard will be measured directly and linearly with its volume. Thus, the buffer capacity of 100 ml is 1 / 10 of the buffer capacity of 1.00 liters, and the buffer capacity of 10 ml is 1 / 100 of the buffer capacity of 1.00 liters. As a result, the volume of the standard can be conveniently adjusted and then standardized back to 1 liter as desired.

[0163] It is not always convenient to make the above 10 mM acetate buffer capacity standard for practical use. However, similar to acetate buffer, other various buffer capacity standards can be made and used using various other buffering agents. Provided only that the buffer standards are properly prepared, these standards can be calibrated against the above acetate buffer standard and then used in the field. The results obtained using such substitute standards can then be expressed in terms of the above acetate standard without substantial distortion or error.

[0164] The buffer capacity of such a surrogate standard can also be calibrated by calculation. To do so, the buffer capacity of the surrogate standard is measured directly and expressed in mEq / unit volume / pH. Measurements based on the surrogate standard can then be normalized to the acetate standard using the ratio of the buffer capacity of the surrogate standard to the buffer capacity of the acetate standard, expressed in mEq / unit volume / pH.

[0165] Using such methods commonly utilized in metrology for relating actual standards back to reference standards, the acetate buffer standards described above provide a portable, measurable, reliable and accurate standard for the measurement of the buffering capacity of any composition, and one that can be easily compared to different standards applied to other compositions using similar methods.

[0166] b. Preparation of buffer capacity standards Buffer capacity standards can be prepared using established methods in analytical chemistry. See, e.g., ANALYTICAL CHAMISTRY, 3rd ed., Douglas A. Skoog and Donald M. West, Holt, Rinehart and Winston, New York (1979), particularly the methods described in Chapter 9 (pp. 186-226), Chapter 10 (pp. 227-233), and pages 583-588; TEITZ TEXTBOOK OF CLINICAL CHEMISTRY, 3rd ed., Burtis and Ashwood, eds., WB Saunders Company, Philadelphia, PA (1999), particularly Chapter 1 and Tables 50-13 to 50-16 on general laboratory techniques for buffer preparation and calibration; THE TOOLS OF BIOCHEMISTRY, Terrance G. Cooper, John Wiley & Sons, New York, NY (1977), particularly Chapter 1, pp. 1-35, and also Tables 1-3, 1-4, and 1-5 and the text therein; PROTEIN PURIFICATION PRINCIPLES AND PRACTICE, 3rd Ed., Robert K. Scopes, Springer-Verlag, New York, NY (1994), especially pages 160-164, especially Tables 6.4 and 6.5 therein and the text thereabout, Chapter 12, Section 3, pages 324-333 therein, especially Tables 12-4 and 12-5 therein and the text thereabout, and the entirety of Appendix C: Buffers for Use in Protein Chemistry; and REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21st Ed., Beringer et al., Eds., Lippincott, Williams & Wilkins, Philadelphia, PA (2005), especially the portions concerning buffering agents, buffer solutions, buffer capacity, etc., each of which is incorporated herein by reference in its entirety, particularly with respect to the preparation and use of buffers and buffer capacity standards according to the present invention.

[0167] The water used in the preparation of the buffer capacity standards must be highly purified, preferably Type I Water, such as milliQ water, or triple distilled water. The buffer reagents must be pure and in particular free of any substances that may alter the pH or buffer capacity of the standards, such as reference grade or ACS grade reagents suitable for applications requiring analytical chemical analysis, as described in the above references, in particular TEITZ and REMINGTON cited above, the contents of which are incorporated herein by reference in their entirety, especially in the sections relating to analytical grade water and reagents.

[0168] The exact composition of the buffer reagents should be well established. For each buffer reagent, its molecular weight should be precisely known. The above molecular weight should be for the reagent to be used and should include the weight of any adduct compounds, such as hydrates, contained in this reagent. The effective number of hydrogen donors or hydrogen acceptors per molecule should be precisely known for each buffer reagent. The proportional distribution of various forms, such as hydrates, should be known for each reagent, including mixtures of such forms. The concentration of liquid buffer reagents should be precisely known, preferably in moles / volume and moles / mass (e.g., moles / liter and moles / gm or kg). Hygroscopic substances should be dried to remove moisture so that the reagents can be weighed out accurately.

[0169] Generally speaking, the information provided by major suppliers of reagents and reference grade reagents is sufficiently accurate for the preparation of the above buffer capacity standards, and well-known standard techniques routinely used in analytical chemistry can be used to dry "hygroscopic materials" so that the buffer capacity standards can be accurately quantified.

[0170] As described in the above references, well-established and routinely used analytical chemistry methods can be used to prepare and calibrate buffer capacity standards as well as acid and base solutions such as 1N HCl and 1N NaOH (to name just two) for titrating sample protein solutions to measure buffer capacity. It should be noted that the preparation of NaOH solutions for titration must be done in a manner that eliminates errors resulting from the interaction of certain dissolved gases with the basic solution and the effect of this solvation on the pH change. See, for example, Skoog and West (1979) and other references cited above regarding the preparation and calibration of buffers and buffer standards. The contents of the above references are incorporated herein by reference in their entirety, particularly in the section above regarding the preparation of titration standards.

[0171] c. Empirical criteria for buffer capacity The titration of standards and samples to measure buffer capacity can be performed using well-known, routine methods. The titration can be performed manually. The titration can also be performed using an automatic titrator. A wide range of automatic titrators suitable for use with the present invention in this regard are commercially available from a number of vendors. Methods suitable for use with the present invention in this regard are the same as those described in the references cited above for the preparation and calibration of buffer standards, each of which is incorporated herein by reference in its entirety, particularly in the portions relating to the titration of known and unknown solutions to measure buffer capacity.

[0172] 2. Buffering by proteins and their buffering capacity a. Measurement of hydrogen equilibrium and buffer capacity of proteins Proteins always contain many acidic and basic components. As a result, the hydrogen ion equilibrium of a protein is extremely complex. In fact, a complete description of the hydrogen ion equilibrium of a protein in a given environment is beyond current theoretical and computational methods. Therefore, empirical measurement of protein buffer capacity is preferred. Methods developed, available, and routinely used by the skilled artisan for accurate empirical measurement of protein hydrogen equilibrium are well suited for measuring the buffering properties of proteins for the development of self-buffering protein formulations of the present invention. Thus, the pH titration curve of a protein can be measured according to the present invention by well-known methods (e.g., the methods described and exemplified in the pH titration study of ribonuclease by Tanford and coworkers). See T. Shedlovsky (ed.), ELECTROCHEMISTRY IN BIOLOGY AND MEDICINE, John Wiley and Sons, New York, 1955, Chapter 13; C. Tanford and J. D. Hauenstein, J. Am. Chem. Soc. 78, 5287 (1956); C. Tanford, PHYSICAL CHEMISTRY OF MACROMOLECULES, John Wiley and Sons, New York, 1961, especially pages 554-567. All of the above references are incorporated herein by reference, especially those sections relating to hydrogen ion titration of proteins and the determination of protein buffering action and buffering capacity.

[0173] However, the present invention does not require precise measurements as described in the above references. Rather, the buffering properties and buffering capacities of the proteins of the present invention can be measured using methods described in standard references on analytical chemistry and biochemistry, such as Skoog (1979), Cooper (1977), and Scopes (1994), cited above, all of which are incorporated herein by reference in their entirety, particularly with respect to the empirical determination of titration curves for proteins within the given pH ranges of the present invention.

[0174] The determination of titration curves and buffering capacity according to the present invention is described in detail in the Examples below for acetate buffers and various pharmaceutical proteins. Thus, the pH titration curves of proteins can be empirically determined over a specific limited pH range for a given formulation according to the methods described in the above references. These methods are in many ways the same as those used in analytical chemistry for the titration of small molecules such as acetate buffers (as shown in the Examples). However, some great care must be taken in handling proteins to maintain the conformation and function required for an effective formulation.

[0175] Protein titration can be performed manually or using an automated titrator. Manual titration equipment and automated titrators are readily available from a number of suppliers and vendors. Suitable methods for measuring protein pH titration curves and buffer capacity are illustrated in the Examples by reference to titration of acetate buffer standards over a defined pH range and titration of several different therapeutic proteins. These methods can be used to measure the elementary ionization behavior and buffer capacity of any other protein according to the present invention.

[0176] A particular aspect of the invention is to measure the buffer capacity of a protein as a function of its concentration in solution. In a preferred method in this regard, solutions of a given protein are prepared at a series of graded concentrations. A pH titration curve is measured for each concentration of the protein across the pH range of interest. Preferably, the titration curve is measured for the range of interest using both base titrations and acid titrations. In certain preferred embodiments, the above data is plotted on a graph of the equivalents of acid or base added versus the measured pH of each solution. Typically, the titration data for each concentration is closely fitted to a straight line, preferably determined by least squares regression analysis, for a range of about 0.5-1.0 pH units. In a preferred embodiment in this regard, the buffer capacity of the protein at each concentration is equal to the slope of the regression line expressed in units of equivalents / ml / pH (or a fraction thereof). Similarly, also useful in the present invention in this regard is the relationship between the buffer capacity of a protein and the concentration of the protein. In certain preferred embodiments, this relationship is measured by least squares regression analysis of a best-fit line of the buffer capacity data, determined according to the above data plotted on a graph of buffer capacity versus protein concentration.

[0177] The empirical data regarding the buffer capacity of a protein according to the invention preferably relates to the buffer capacity of a standard acetate buffer, i.e. in a particularly preferred embodiment of the invention in this respect, the buffer capacity of a given protein at a given concentration in a given formulation, measured as described above, is equal to the concentration of a standard acetate buffer having an equivalent buffer capacity.

[0178] While the empirical measurements described herein are generally critical aspects of formulating self-buffering compositions according to various aspects and preferred embodiments of the present invention, theoretical and computational methods (in combination with the empirical measurements) can also be productively employed to guide the design, manufacture and use of such compositions, as described below.

[0179] b. Prediction of hydrogen ion equilibrium and buffer capacity of proteins The ionization of hydrogen in proteins is complex, but in general terms can be divided into a pH range dictated by the ionized hydrogen of the amino acid side chains and a pH range dictated by the terminal amino and carboxyl groups. a is typically around 3.1. The pK of the acidic hydrogens in the side chains of aspartic acid and glutamic acid a is approximately 4.4. a is approximately 6.0. The hydrogen ionization pK of the terminal amino group a is typically around 7.5. a is approximately 8.5. The tyrosine hydroxyl and lysine amine both have pK values ​​of approximately 10. a The pK of arginine a is approximately 12.

[0180] Conformational folding generally partitions large polypeptides and proteins in polar solvents into exposed, solvent-accessible regions and a mostly non-polar core region with little or no contact with the surrounding environment. Folding generates many environments between these two extremes. Furthermore, the microenvironment around a given amino acid side chain in a protein is typically influenced by one or more of the following: solvent effects; ionic binding; chelation; complex formation; association with cofactors; and post-translational modifications; to name a few possibilities. Each of these affects the pK of ionization of a given amino acid in a protein. a Thus, the pK a is the pK of the free amino acid a can vary dramatically from

[0181] In fact, the pK of the amino acid in a protein depends on its microenvironment. aFluctuations of have been used to study protein folding and the configuration and charge state of specific amino acids in folded proteins. Several protein titration curves reported by Tanford et al. have in common a complex of several broad features. Typically, only a portion of the ionized protons are accounted for in the titration curve; other ionized protons are apparently located in the core region and inaccessible to the solvent. The pK of individual homologous side chains can sometimes be detected. a These pK a is usually the pK of the free amino acid a Close to.

[0182] The strongest buffering effect of proteins does not usually occur at the isoelectric point, as might be misunderstood. In fact, the buffering is determined by the hydrogens in the amino acid side chains and the terminal hydrogens, and therefore by the pK of the ionized hydrogen in the free amino acid, as shown above. a Of these, the most important for formulating compositions of proteins, particularly certain pharmaceutical proteins, that are more soluble and / or more stable at slightly acidic pH (pH 4-6), are the pK values ​​of the carboxyl hydrogens of the amino acids aspartic acid and glutamic acid. a That is, the buffering effect occurs in the range of pH 4.0 to 5.5, particularly about 4.5.

[0183] There are various methods available for estimating the buffering capacity of a given protein in a given solution at a given pH. The methods range from highly specialized and complex computer models to methods that can be implemented on a manual calculator. None of these methods are perfect or completely accurate. However, they do provide useful estimates in some cases.

[0184] For example, potentially useful knowledge of the buffering capacity of a protein in solution can be obtained from its amino acid composition, the pK of the terminal amino and carboxyl groups, and the hydrogen donors and hydrogen acceptors of the amino acid side chains. a, can be calculated based on the concentration of the protein, as well as the pH of the solution.

[0185] For example, the pK of the side chain carboxyl hydrogen of glutamic acid (as a free amino acid) a A potentially useful estimate of the buffering capacity of a protein at pHs in the range can be obtained from the molecular weight of the protein and the number of glutamic acid residues it contains. Dividing the former by the latter gives the weight per equivalent of glutamic acid, and thus the pK of glutamic acid. a The weight of one equivalent of ionized hydrogen at 1000 . The carboxyl groups in the side chains of glutamic acid and aspartic acid have almost the same pK a Since both pK a To obtain an estimate of the buffer capacity before and after, the results of the above calculations for these two amino acids must be added. a The estimated buffer capacity of a protein solution in can be calculated from the concentration of the protein in solution and the naturally occurring intrinsic factor, i.e., weight per equivalent of ionized hydrogen. Dividing the concentration by the weight per equivalent gives an estimate of the buffer capacity in Eq / volume units. Such estimates are often considered to be too high, because some residues are usually sequestered in regions of the protein that are not accessible to the solvent, and therefore do not contribute to the actual buffer capacity. In certain instances, the above sequestering effects on buffer capacity can be taken into account. For example, a fractional factor reflecting theoretical or empirical estimates of sequestering can be applied to adjust the original calculation.

[0186] Such calculations are generally less practical and will be less accurate than empirical measurement of protein buffering capacity by methods described elsewhere herein, but they do serve to provide a rough maximum estimate of the buffering capacity of a protein in solution.

[0187] 3. protein The invention disclosed herein can be practiced with any protein that provides sufficient buffering capacity at the desired pH range within parameters such as protein concentration required for the desired formulation. Preferred proteins in this regard are pharmaceutical proteins for therapeutic use in animals and / or humans, particularly for therapeutic use in humans. Similarly preferred proteins are proteins that are soluble in aqueous solutions, particularly proteins that are soluble at relatively high concentrations and proteins that are stable over time. Further preferred proteins are proteins that contain a relatively large number of solvent-accessible amino acids with ionization constants of side chain hydrogens close to the pH of the desired buffering.

[0188] Moreover, preferred proteins of the present invention are those for pharmaceutical preparations that do not induce significant adverse antigenic responses after administration to a subject. Preferred in this regard are proteins for medical use in animals and / or humans, and particularly with regard to the latter, humanized and human proteins.

[0189] Furthermore, preferred proteins of the present invention are proteins that selectively bind to specific targets, such as ligand-binding proteins and protein ligands. Antigen-binding proteins, proteins derived therefrom, and proteins related thereto are particularly preferred embodiments of the present invention in this regard. Highly preferred proteins of the present invention in this regard are antibodies and proteins derived from antibodies or proteins incorporating antibodies in whole or in part, such as monoclonal antibodies, polyclonal antibodies, genetically engineered antibodies, hybrid antibodies, bispecific antibodies, single-chain antibodies, genetically modified antibodies (e.g., antibodies with one or more amino acid substitutions, additions, and / or deletions (antibody variants)), chimeric antibodies, antibody derivatives, antibody fragments that can be derived from any of the above antibodies and can also be derivatives of these antibodies that have been similarly engineered or modified, fusion proteins that include antibodies or portions derived from antibodies or antibody fragments (e.g., any of the above antibodies or variants or derivatives of the antibodies), conjugates that include antibodies or portions derived from antibodies (e.g., any of the above antibodies or variants or derivatives of the antibodies), and chemically modified antibodies, antibody fragments, antibody fusion proteins, etc. (e.g., all of the above antibodies).

[0190] a. Antibodies, antibody-derived proteins, antibody-related proteins, etc. Particularly preferred proteins according to the invention are antibody polypeptides, such as heavy and light chain polypeptides, having sequences identical to those occurring in and constituting naturally occurring antibodies (e.g., amino acid sequences occurring in serum and antisera), including, for example, polypeptides and proteins isolated from natural sources, as well as polypeptides and proteins produced by hybridoma technology, activation of endogenous genes (e.g., by homologous or non-homologous recombination), expression of exogenous genes under the control of endogenous transcriptional control regions, expression of exogenous expression constructs, semi-synthesis and de novo synthesis. The above techniques are some of the techniques commonly used for the production of antibodies and antibody-related polypeptides and proteins that can be used to produce the antibody polypeptides and proteins according to the invention.

[0191] These antibody-related polypeptides and proteins include those that have a de novo amino acid sequence in whole or in part, and in particular those that comprise all or one or more portions of an antibody (i.e., a contiguous stretch of amino acids having the same sequence as any four or more residues in the amino acid sequence of a naturally occurring antibody polypeptide), those that have an amino acid sequence that corresponds in some way to the amino acid sequence of a naturally occurring antibody but differs in other ways, those that have an amino acid sequence that is identical to the corresponding amino acid sequence in nature or a sequence related thereto, but differs from the corresponding amino acid sequence as a result of one or more post-translational modifications, and those that are included as part of any of the above antibodies fused (in whole or in part) to one or more polypeptide regions that are from or derived from or related to a second, different antibody polypeptide, and that are from or derived from any other polypeptide or protein, that are similar to but distinct from the antibody-related polypeptides and proteins of the invention, whether naturally occurring or not, and that have semi-de novo amino acid sequences and / or de novo amino acid sequences.

[0192] Further, the preferred protein of the present invention described herein is all the above modified proteins.Such modified proteins include the protein that is chemically modified by non-covalent, covalent, or both covalent and non-covalent bonds.Similarly, it includes all the above proteins that further comprise one or more post-translational modifications that can be made by cell modification system, or the modification that is introduced ex vivo by enzymatic and / or chemical methods, or the modification that is introduced by other methods.

[0193] A preferred protein of the invention in this regard is a typical dimer (LH) 2Fab fragments as produced by cleaving antibodies with a specific protease that cleaves the heavy chains "at" the disulfide bonds that link them between the variable region and the adjacent constant region, while leaving the light chains intact. This cleavage releases an Fc fragment containing the remaining part of the linked heavy chain, and two dimeric Fab fragments, each containing an intact light chain and a heavy chain variable region. Fab fragments can also be produced by other techniques that do not require the isolation of native antibodies and / or cleavage with a protease.

[0194] Also preferred are Fab fragments, which are produced using a protease that cleaves "between or below" disulfide bonds in much the same way as Fab fragments. 2 As a result, the two Fab fragments are linked by disulfide bonds to form a single Fab 2 Released as fragments: Fab 2 Fragments can be produced by many other techniques, including techniques that do not require isolation of intact antibodies or cleavage with a protease having the required specificity. Additionally, monospecific Fab fragments can be produced by a number of techniques, including techniques that do not require isolation of intact antibodies or cleavage with a protease having the required specificity. 2 Fragments and bispecific Fabs 2 Both fragments can now be produced by a variety of routine techniques.

[0195] An equally preferred protein in this regard is an engineered antibody fragment, Fab, in which three Fab fragments are linked together. 3 It is a fragment. Fab 3 The fragments may be monospecific, bispecific, or trispecific and may be produced in a variety of ways well known to those of skill in the relevant art.

[0196] Other preferred proteins in this regard are Fc fragments, e.g., Fab fragments or Fab 2The Fc fragment is produced by cleavage with a protease in the same manner as used to produce either of the fragments. However, for the production of the Fc fragment, the dimeric heavy chain containing fragment is isolated rather than the light chain containing fragment. The Fc fragment lacks the antigen binding site, but contains an effector region that plays a role in physiological processes involving antibodies. The Fc fragment can be produced by a variety of techniques well known and routinely used by those skilled in the art for this purpose.

[0197] Other preferred proteins in this regard are single chain variable fragments ("scFv"). scFvs are fusion proteins made by combining the variable regions of heavy and light immunoglobulin chains. The heavy and light chains of scFvs are typically joined by a short serine, glycine linker. scFvs have the same specificity as the antibodies from which they are derived. scFvs were originally produced by phage display techniques, but can now be made by a variety of well-known methods.

[0198] Also preferred is Bis-scFv, which is a fusion of two scFvs. Bis-scFvs can be monospecific or bispecific. A variety of methods are well known and can be applied to the production of Bis-scFvs of the present invention.

[0199] Also preferred according to the invention in this respect are minibodies; monospecific and bispecific dimers; monospecific, bispecific and trispecific triabodies; monospecific, bispecific, trispecific, tetraspecific tetrabodies; VhH domains, V-NAR domains, V H Domain, V L domain, camelid Ig, Ig NAR, and other antibodies.

[0200] Similarly, preferred embodiments according to the various aspects and preferred embodiments of the invention in this and other respects are proteins comprising one or more CDRs and / or CDR-derived and / or CDR-related regions of an antibody, or one or more FRs and / or FR-derived and / or FR-related regions of an antibody. In this regard, CDR refers to the complementary determining region, i.e., the hypervariable region of the light or heavy chain of an antibody, typically about 9-12 amino acids in length that is usually the significant portion of the antigen-specific binding portion of an antibody. In this regard, FR refers to the framework region of an antibody, i.e., the region of about 15-20 amino acids that separates the CDRs in the antigen-specific binding portion of an antibody.

[0201] The terms CDR-derived and CDR-related, and FR-derived and FR-related, have the same meaning as CDR and FR, respectively, as explained for the terms antibody-derived and antibody-related in relation to the term antibody in the "Terminology" section above.

[0202] For antibodies, antibody-derived and antibody-related proteins according to these and other aspects of the invention disclosed herein, see, for example, Protein Engineering: Principles and Practice, Jeffrey L. Cleland and Charles S. Craik, eds., Wiley-Liss, Inc., New York (1996), in particular Kelley, Robert F., "Engineering Therapeutic Antibodies," Chapter 15, pp. 399-434, and Hollinger, P. & Hudson, P., "Engineered antibody fragments and the rise of single domains," Nature Biotechnology, September 2005, pp. 1126-1136. Each of these references is incorporated herein by reference in its entirety, particularly in the sections relating to the structure and operation of antibodies (particularly biopharmaceutical antibodies) and antibody-derived and antibody-related proteins (particularly the antibody-derived and antibody-related pharmaceutical proteins of the invention disclosed herein).

[0203] Of all the above proteins, particularly preferred herein are human, humanized, and other proteins that do not provoke a significant adverse immune response when administered to humans.Also preferred herein are all the above proteins that similarly do not provoke a significant adverse immune response when administered to non-humans.

[0204] Very particularly preferred proteins according to the invention are fusion proteins comprising antibodies and / or antibody-derived proteins, polypeptides, or fragments etc., including all of the proteins mentioned above. In this regard, very particularly preferred fusion proteins of the invention are fusion proteins comprising an antibody or antibody-derived protein or fragment as described above and a ligand-binding moiety, as specifically described herein.

[0205] b. Target-binding proteins Similarly, preferred proteins of the invention in this regard are antibodies and other types of target binding proteins, and proteins related thereto or derived therefrom, as well as protein ligands, and proteins related thereto or derived therefrom. Particularly preferred ligand binding proteins in this regard are proteins that bind to signal proteins and effector proteins, and proteins related thereto or derived therefrom.

[0206] Such binding proteins, such as antibodies, proteins derived therefrom and related proteins, are binding proteins that bind to one or more of the following proteins, either alone or in any combination: (i) CD proteins, including but not limited to, CD3, CD4, CD8, CD19, CD20, and CD34; (ii) HER receptor family proteins, e.g., HER2, HER3, HER4, and EGF receptor; (iii) cell adhesion molecules, e.g., LFA-1, Mol, p150, 95, VLA-4, ICAM-1, VCAM, and αv / β3 integrin; (iv) growth factors, such as, but not limited to, vascular endothelial growth factor ("VEGF"); growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, Mullerian inhibitory substance, human macrophage inflammatory protein (MIP-1-α), erythropoietin (EPO), nerve growth factor (e.g., NGF-β), platelet derived growth factor (PDGF), fibroblast growth factor (e.g., aFGF and bFGF), epidermal growth factor (EGF), transforming growth factor (TGF) (e.g., TGF-α and TGF-β, particularly TGF-β (e.g., TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5)), insulin-like growth factor I and II (IGF-I and IGF-II), des(1-3)-IGF-I (brain IGF-I), and bone morphogenetic factors; (v) insulin and insulin-related proteins, including, but not limited to, insulin, insulin A chain, insulin B chain, proinsulin, and insulin-like growth factor binding protein; (vi) aggregation and aggregation-related proteins, such as, inter alia, factor VIII, tissue factor, von Willebrand factor, protein C, alpha-1-antitrypsin, plasminogen activators (e.g., urokinase and tissue plasminogen activator ("t-PA")), bombadin, thrombin, and thrombopoietin; (vii) colony stimulating factors (CSFs), such as the following, in particular M-CSF, GM-CSF, and G-CSF; (viii) other blood and serum proteins, including but not limited to albumin, IgE, and blood group antigens; (ix) receptors and receptor-associated proteins, such as the flk2 / flt3 receptor, the obesity (OB) receptor, the growth hormone receptor, and the T cell receptor; (x) neurotrophic factors, such as, but not limited to, bone induced neurotrophic factor (BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6); (xi) relaxin A chain, relaxin B chain, and prorelaxin; (xii) Interferons, e.g., interferon alpha, beta, and gamma; (xiii) interleukins (ILs), e.g., IL-1 to IL-10; (xiv) Viral antigens, including but not limited to, AIDS envelope virus antigens; (xv) lipoproteins, calcitonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factors α and β, enkephalinase, RANTES (normally expressed and secreted by T cells and regulated upon activation), mouse gonadotropin-related peptide, deoxyribonuclease (Dnase), inhibin, and activin; (xvi) integrins, proteins A or D, rheumatoid factors, immunotoxins, bone morphogenetic proteins (BMPs), superoxide dismutase, surface membrane proteins, decay accelerating factors (DAFs), AIDS envelopes, transport proteins, homing receptors, addressins, regulatory proteins, immunoadhesins, antibodies; and (xvii) A biologically active fragment or variant of any of the above.

[0207] In all of the above, particularly preferred are proteins that are effective therapeutic agents, particularly proteins that exert a therapeutic effect by binding to a target (including, in particular, the target proteins listed above, targets derived from these, targets related to these, and modifications thereof).

[0208] c. Certain specific proteins Certain specific proteins are the particular antibodies and antibody-related proteins, such as peptide antibodies, listed immediately below and elsewhere herein.

[0209] OPGL-specific antibodies and peptide antibodies, etc. (also called RANKL-specific antibodies, peptide antibodies, etc.) include, for example, fully humanized antibodies and human OPGL-specific antibodies, particularly fully humanized monoclonal antibodies, such as, but not limited to, antibodies described in International Publication No. WO 03 / 002713, the contents of which are incorporated herein in their entirety with respect to OPGL-specific antibodies and antibody-related proteins, particularly OPGL-specific antibodies and antibody-related proteins having the sequences set forth in this publication, particularly, but not limited to, OPGL-specific antibodies having any of the sequences 9H7; 18B2; 2D8; 2E11; 16E1; and 22B3 set forth in this publication, such as the light chain of SEQ ID NO: 2 set forth in FIG. 2 and / or the heavy chain of SEQ ID NO: 4 set forth in FIG. 4 of this publication. Each OPGL-specific antibody and peptide antibody, etc. is individually and specifically incorporated herein by reference in its entirety as fully disclosed in the above publication. Acid and base titrations of OPGL-specific antibody ("Ab-hOPGL") over the pH ranges 4.5-5.0 and 5.0-5.5 are described in the Examples below. Calculations of the buffer capacity of Ab-hOPGL in these pH ranges are also described in the Examples below.

[0210] Myostatin binding agents or peptide antibodies include, for example, myostatin-specific peptide antibodies, particularly those described in U.S. Patent Application Publication No. 2004 / 0181033, the contents of which are incorporated herein by reference in their entirety in the portions relating to myostatin-specific peptide antibodies, including, but not limited to, the mTN8-19 family of peptide antibodies (e.g., peptide antibodies of SEQ ID NOs: 305-351, including TN8-19-1-TN8-19-40, TN8-19 con1 and TN8-19 con2); the mL2 family of peptide antibodies of SEQ ID NOs: 357-383; the mL15 family of SEQ ID NOs: 384-409; the mL17 family of SEQ ID NOs: 410-438; the mL20 family of SEQ ID NOs: 439-446; the mL21 family of SEQ ID NOs: 447-452; the mL24 family of SEQ ID NOs: 453-454; and the peptide antibodies of SEQ ID NOs: 615-631. Each myostatin binding agent or myostatin binding peptide antibody is individually and specifically incorporated by reference herein in its entirety as fully disclosed in the above publications.

[0211] IL-4 receptor-specific antibodies, particularly IL-4 receptor-specific antibodies that inhibit the activity of IL-4 and / or IL-13 mediated by binding to the receptor, include, for example, the IL-4 receptor-specific antibodies described in International Patent Application No. PCT / US2004 / 03742, International Publication No. WO 2005 / 047331. The contents of this document are incorporated herein by reference in their entirety in the portion relating to IL-4 receptor-specific antibodies, particularly those IL-4 receptor-specific antibodies described therein, including, but not limited to, the IL-4 receptor-specific antibodies designated therein: L1H1; L1H2; L1H3; L1H4; L1H5; L1H6; L1H7; L1H8; L1H9; L1H10; L1H11; L2H1; L2H2; L2H3; L2H4; L2H5; L2H6; L2H7; L2H8; L2H9; L2H10; L2H11; L2H12; L213; L2H14; L3H1; L4H1; L5H1; L6H1. Each IL-4 receptor-specific antibody is individually and specifically incorporated herein by reference in its entirety as fully disclosed in the above publication. Acid and base titrations of an IL-4 receptor specific antibody ("Ab-hIL4R") over the pH ranges 4.5-5.0 and pH 5.0-5.5, and calculation of buffer capacity in this range are described in the Examples below.

[0212] Interleukin 1-receptor 1 ("IL1-R1") specific antibodies, peptide antibodies and related proteins, etc., include, for example, but are not limited to, the IL1-R1 specific antibodies described in U.S. Patent Application Publication No. 2004 / 097712A1, the contents of which are incorporated herein by reference in their entirety in the portions relating to IL1-R1 specific binding proteins, particularly monoclonal antibodies, particularly, but not limited to, the monoclonal antibodies designated therein: 15CA, 26F5, 27F2, 24E12, and 10H7. Each interleukin 1-receptor 1 specific antibody is individually and specifically incorporated herein by reference in its entirety as fully disclosed in the above U.S. Patent Application Publication.

[0213] Examples of Ang2-specific antibodies and peptide antibodies and related proteins include, but are not limited to, the Ang2-specific antibodies and peptide antibodies and related proteins described in International Publication No. WO 03 / 057134 and U.S. Application Publication No. US2003 / 0229023. The contents of each document are incorporated herein by reference in their entirety in the sections relating to Ang2-specific antibodies and peptide antibodies, in particular Ang2-specific antibodies and peptide antibodies of the sequences described therein, for example, but not limited to: L1(N); L1(N) WT; L1(N) 1K WT; 2xL1(N); 2xL1(N) WT; Con4 (N), Con4 (N) 1K WT, 2xCon4 (N) 1K; L1(C); L1(C) 1K; 2xL1 (C); Con4 (C); Con4 (C) 1K; 2xCon4 (C) 1K; Con4-L1 (N); Con4-L1 (C); TN-12-9 (N); C17 (N); TN8-8 (N); TN8-14 (N); Con 1 (N). Similarly, Ang2-specific antibodies and peptide antibodies and related proteins, etc., include anti-Ang2 antibodies and preparations thereof, for example, WO 2005 / 023666. and Ab546; A551; Ab553; Ab555; Ab558; Ab559; Ab565; AbF1AbFD; AbFE; AbFJ; AbFK; AbG1D4; AbGC1E8; AbH1C12; AblA1; AblF; AblKAblP; and AblP in various orders as described in this document. Each Ang2-specific antibody and peptide antibody and related proteins is individually and specifically incorporated by reference herein in its entirety as fully disclosed in the above publications.

[0214] NGF-specific antibodies include, for example, particularly, but not limited to, the NGF-specific antibodies described in US Patent Publication No. US2005 / 0074821.The contents of this document are incorporated herein by reference in their entirety, particularly, but not limited to, the portions relating to NGF-specific antibodies and related proteins in this regard, particularly, but not limited to, the NGF-specific antibodies 4D4, 4G6, 6H9, 7H2, 14D10 and 14D11 designated therein.Each NGF-specific antibody is individually and specifically incorporated herein by reference in its entirety as fully disclosed in the above publication.

[0215] CD22-specific antibodies and related proteins include, for example, those described in U.S. Patent No. 5,789,554, the contents of which are incorporated herein by reference in their entirety with respect to CD22-specific antibodies and related proteins, particularly human CD22-specific antibodies, including, but not limited to, humanized and fully human antibodies, including, but not limited to, humanized and fully human monoclonal antibodies, particularly, but not limited to, human CD22-specific IgG antibodies, such as, for example, disulfide-linked dimers of human-mouse monoclonal hLL2 gamma and human-mouse monoclonal hLL2 kappa chains (including, but not limited to, the human CD22-specific fully humanized antibody in Epratuzumab, CAS Registry No. 501423-23-0). As an example of the present invention, acid and base titrations of CD22-specific antibodies ("Ab-hCD22") over the pH ranges 4.5-5.0 and pH 5.0-5.5 are described in the Examples below.

[0216] IGF-1 receptor-specific antibodies and related proteins include, for example, those described in International Patent Application No. PCT / US2005 / 046493, the contents of which are incorporated herein by reference, including, but not limited to, the IGF-1 receptor-specific antibodies and related proteins designated therein, including, but not limited to, the IGF-1 specific antibodies L1H1, L2H2, L3H3, L4H4, L5H5, L6H6, L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, L14H14, L15H15, L16H16, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23, L24H24, L25H26, L26H28, L27H29, L28H30, L29H31, L32H32, L33H33, L34H34, L35H36, L36H37, L37H38, L38H39, L39H40, L39H41, L39H42, L39H43, L39H44, L39H45, L39H46, L39H47, L39H48, L39H49 ...9, L39H49, L39H49, L39H49, L39H49, L With respect to 25H25, L26H26, L27H27, L28H28, L29H29, L30H30, L31H31, L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39, L40H40, L41H41, L42H42, L43H43, L44H44, L45H45, L46H46, L47H47, L48H48, L49H49, L50H50, L51H51 and L52H52, each of which is individually and specifically incorporated by reference herein in its entirety as fully disclosed in the above-referenced international applications.

[0217] B-7 related protein 1 ("B7RP-1") specific antibodies include, in particular, B7RP-specific fully human monoclonal IgG2 antibodies (B7RP-1 is also referred to in the literature as B7H2, ICOSL, B7h, and also as CD275), in particular fully human IgG2 monoclonal antibodies which bind to an epitope in the first immunoglobulin-like domain of B7RP-1, in particular fully human IgG2 monoclonal antibodies which inhibit the interaction of B7RP-1 with its natural receptor, in particular ICOS on activated T cells, and in particular, in all respects, the B7RP-1 specific antibodies disclosed in U.S. Provisional Application No. 60 / 700,265, filed July 8, 2005. The contents of this document are incorporated herein by reference in their entirety with respect to such antibodies and related proteins, including, but not limited to, the following B7RP-1 specific antibodies designated therein: 16H (having light and heavy chain variable region sequences SEQ ID NO:1 and SEQ ID NO:7, respectively); 5D (having light and heavy chain variable region sequences SEQ ID NO:2 and SEQ ID NO:9, respectively); 2H (having light and heavy chain variable region sequences SEQ ID NO:3 and SEQ ID NO:10, respectively); 43H (having light and heavy chain variable region sequences SEQ ID NO:6 and SEQ ID NO:14, respectively); 41H (having light and heavy chain variable region sequences SEQ ID NO:5 and SEQ ID NO:13, respectively); and 15H (having light and heavy chain variable region sequences SEQ ID NO:4 and SEQ ID NO:12, respectively). Each B7RP-1 specific antibody and related proteins is individually and specifically incorporated herein by reference in its entirety as fully disclosed in the above-referenced U.S. provisional application. Acid and base titrations and buffer capacity measurements of B7RP-1 specific antibody ("Ab-hB7RP1") are shown in the Examples below.

[0218] IL-15 specific antibodies, peptide antibodies and related proteins include, for example, particularly humanized monoclonal antibodies, particularly the antibodies disclosed in U.S. Patent Publication Nos.: US2003 / 0138421; US2003 / 023586; US2004 / 0071702. Each IL-15 specific antibody, etc. is incorporated herein by reference in its entirety with respect to IL-15 specific antibodies and related proteins, e.g., peptide antibodies, particularly, e.g., HuMax IL-15 antibodies and related proteins, such as, but not limited to, 146B7.

[0219] IFNγ-specific antibodies, particularly human IFNγ-specific antibodies, in particular fully human anti-IFNγ antibodies, include, for example, the IFNγ-specific antibodies described in US Patent Publication No. US2005 / 0004353. The contents of this document are incorporated herein by reference in their entirety, including without limitation, the IFNγ-specific antibodies, in particular the antibodies designated in this document, e.g., 1118;1118 * ;1119;1121;and 1121 * Each IFNγ-specific antibody is individually and specifically incorporated by reference herein in its entirety as fully disclosed in the above-referenced U.S. Patent Publication.

[0220] TALL-1 specific antibodies and other TALL specific binding proteins include, for example, those described in US Application Publication No. 2003 / 0195156, the contents of which are incorporated herein by reference in their entirety with respect to TALL-1 binding proteins, particularly the molecules in Tables 4 and 5B. Each TALL-1 specific antibody and other TALL specific binding protein is individually and specifically incorporated herein by reference in its entirety as fully disclosed in the above US Application Publication.

[0221] Stem cell factors ("SCF") and related proteins include, for example, the SCF and related proteins described in U.S. Patent Nos. 6,204,363 and 6,207,802. Each SCF and related protein is incorporated herein by reference in its entirety with respect to stem cell factors and related proteins, particularly, for example, stem cell factor "STEMGEN®."

[0222] Flt3-Ligand, ("Flt3L") and related proteins include, for example, Flt3L and related proteins as described in US Pat. No. 6,632,424, the contents of which are incorporated herein by reference with respect to Flt3-Ligand and related proteins.

[0223] IL-17 receptors and related proteins ("IL-17R") include, for example, the IL-17R and related proteins described in U.S. Pat. No. 6,072,033, the contents of which are incorporated herein by reference with respect to Flt3-ligand and related proteins.

[0224] Etanercept, also known as Embrel, and related proteins.

[0225] Actimmune (interferon-gamma-1b), Activase (alteplase), Aldurazme (laronidase), Amevive (alefacept), Avonex (interferon beta-1a), BeneFIX (nonacog alfa), Beromun (tasonermin), Beatseron (interferon beta-1b), BEXXAR (tositumomab), Tev-Tropin (somatropin), Bioclate or RECOMBINATE (recombinant), CEREZME (imiglucerase), ENBREL (etanercept), Eprex (epoetin alfa), EPOGEN / Procit (epoetin alfa), FABRAZYME (agalsidase beta), Fasturtec / Elitek ELITEK (rasburicase), FORTEO (teriparatide), GENOTROPIN (somatropin), GlucaGen (glucagon), Glucagon (glucagon, rDNA derived), GONAL-F (follitropin alfa), KOGENATE FS (ostocog alfa), HERCEPTIN (trastuzumab), HUMATROPE (somatropin), HUMIRA (adalimumab), insulin in solution, INFERGEN® (interferon alfacon-1), KINERET® (anakinra), Kogenate FS (antihemophilic factor), LEUKIN (sargramostim recombinant human granulocyte-macrophage colony-stimulating factor (rhuGM-CSF)), CAMPATH (alemtuzumab), RITUXAN® (rituximab), TNKase (tenecteplase), MYLOTARG (gemtuzumab) ozogamicin), NATRECOR (nesiritide), ARANESP (darbepoetin alfa), NEULASTA (pegfilgrastim), NEUMEGA (oprelvekin), NEUPOGEN (filgrastim), NORDITROPIN CARTRIDGES (somatropin), NOVOSEVEN (eptacog alfa), NUTROPIN AQ (somatropin), Oncaspar (pegaspargase), ONTAK (denileukin difitox), ORTHOCLONEOKT (muromonab-CD3), OVIDREL (choriogonadotropin alfa), PEGASYS (peginterferon alfa-2a), PROLEUKIN (aldesleukin), PULMOZYME (dornase alfa), Retavase (reteplase), REBETRON combination therapy including REBETOL® (ribavirin) and INTRON® A (interferon alfa-2b), REBIF (interferon beta-1a), REFACTO (antihemophilic factor), REFLUDAN (lepirudin), REMICADE (infliximab), REOPRO (abciximab) and ROFERO N®-A (interferon alfa-2a), SIMULECT (basiliximab), SOMAVERT (pegvisomant), SYNAGIS® (palivizumab), Stemben (ansestim, stem cell factor), THYROGEN, INTRON® A (interferon alfa-2b), PEG-INTRON® (peginterferon alfa-2b), XIGRIS® (activated drotrecogin alfa), XOLAIR® (omalizumab), ZENAPAX® (daclizumab), and ZEVALIN® (ibritumomab tiuxetan).

[0226] d. Sequence Variation Particularly preferred proteins for all of the above and below proteins include those which contain a region whose amino acid sequence is at least 70%, particularly at least 80%, very particularly at least 90%, even more particularly at least 95%, particularly at least 97%, very particularly at least 98%, and even very particularly at least 99% identical to a reference amino acid sequence of the above-mentioned binding proteins, especially pharmaceutical binding proteins, e.g. a GenBank or other reference sequence of the reference protein.

[0227] In this regard, identity can be measured using various well-known and readily available amino acid sequence analysis software. Preferred software includes, for example, software that implements the Smith-Waterman algorithm, which is considered to be a satisfactory solution to the problem of searching and aligning sequences. Other algorithms can also be used, especially when speed is an important consideration. Programs that are commonly used for alignment and homology matching of DNA, RNA, and polypeptides and can be used in this regard include FASTA, TFASTA, BLASTN, BLASTP, BLASTX, TBLASTN, PROSRCH, BLAZE, and MPSRCH. The latter implements the Smith-Waterman algorithm implemented on a massively parallel processor created by MasPar.

[0228] The BLASTN, BLASTX, and BLASTP programs are suitable for such determination, the former for comparing polynucleotide sequences and the latter two for comparing polypeptide sequences: BLASTX for comparing polypeptide sequences predicted from all three reading frames of a polynucleotide sequence, and BLASTP for comparing a single polypeptide sequence.

[0229] BLAST offers a variety of user-definable parameters that are set before performing a comparison. Some of these parameters are more visible than others on the graphical user interface provided by NCBI BLAST and other sequence alignment programs accessible on the Internet. The parameter settings and values ​​are presented and explained on the service website and are described and presented in detail in various readily available textbooks, including but not limited to BIOINFORMATICS: SEQUENCE AND GENOME ANALYSIS, 2nd ed., David W. Mount, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (2004), particularly chapters 3, 4, 5, and 6 on general protein and nucleic acid sequence comparisons and BLAST searches; SEQUENCE ANALYSIS IN A NUTSHELL: A GUIDE TO COMMON TOOLS AND DATABASES, Scott Markel and Darryl Leon, O'Reilly & Associates, Sebastopol, California (2003), particularly chapter 7 on BLAST. Each of these references is incorporated herein by reference in its entirety, particularly in the sections relating to the comparison of nucleotide and polypeptide sequences and determining the degree of identity, similarity, homology and / or homology of these sequences, particularly the comparison of these to calculate the degree (percent) of identity between a test sequence and a reference sequence.

[0230] In a preferred embodiment of the invention in this regard, sequence relatedness is defined as the percent identity score returned by any of the various above BLAST comparison searches (with expectation e=10 and all other parameters set to their default values ​​on the NCBI web server) as explained in SEQUENCE ANALYSIS IN A NUTSHELL: A GUIDE TO COMMON TOOLS AND DATABASES, Scott Markel and Darryl Leon, O'Reilly & Associates, Sebastopol, California (2003), pp. 47-51, which is incorporated herein by reference in its entirety and in its entirety with respect to the preferred settings of the parameters of the invention for sequence comparison using BLAST (e.g., sequence comparison on NCBI BLAST).

[0231] The following references provide further information regarding sequence comparison in this and other respects: GUIDE TO HUMAN GENOME COMPUTING, edited by Martin J. Bishop, Academic Press, Harcourt Brace & Company Publishers, New York (1994), which is incorporated herein by reference in its entirety, above, particularly in the portion relating to determining identity and / or homology of amino acid or polynucleotide sequences, particularly Chapter 7. The BLAST program is described in Altschul et al., "Basic Local Alignment Research Tool," J Mol Biol 215: 403-410 (1990), which is incorporated herein by reference in its entirety.Further information on sequence analysis and the determination of homology and identity can be found in many other references well known and readily available to those skilled in the art: NUCLEIC ACID AND PROTEIN SEQUENCE ANALYSIS: A PRACTICAL APPROACH, edited by MJ Bishop and CJ Rawings, IRL Press, Oxford, UK (1987); PROTEIN STRUCTURE: A PRACTICAL APPROACH, edited by TE Creighton, IRL Press, Oxford, UK (1989); Doolittle, RF: "Searching through sequence databases," Met Enz. 183: 99-110 (1990); Meyers and Miller: "Optimal alignments in linear space" Comput. Applica. in Biosci 4: 11-17 (1988); Needleman and Wunsch: "A general method applicable to the search for similarities in amino acid sequence of two proteins," J Mol Biol 48: 443-453 (1989). (1970), and Smith and Waterman "Identification of common molecular subsequences," J Mol Biol 147: 1950 et seq. (1981), each of which is incorporated by reference in its entirety above, particularly with respect to portions relating to sequence comparison and determining identity and homology.

[0232] In particular preferred embodiments in this regard, the activity is 50% to 150% of the reference protein. In particularly highly preferred embodiments, the activity is 60% to 125% of the reference protein, in even more highly preferred embodiments, the activity is 75% to 110% of the reference protein, in even more highly preferred embodiments, the activity is 85% to 125% of the reference, and in even more highly preferred embodiments, the activity is 90% to 110% of the reference.

[0233] 4. formulation Many reagents and methods conventionally used in the formulation of protein pharmaceuticals can be used to formulate self-buffering protein compositions according to the various aspects and preferred embodiments of the present invention. However, in the self-buffering protein formulations of the present invention, buffering is provided substantially entirely by the protein itself, rather than from a buffering agent as in conventional formulations. Moreover, the self-buffering protein formulations according to the various aspects and preferred embodiments of the present invention are substantially free of such buffering agents.

[0234] In many other respects, however, the self-buffering protein compositions according to various aspects and embodiments of the present invention can be formulated using reagents and methods conventionally used in formulating proteins, in particular those used in formulating pharmaceuticals (e.g., pharmaceuticals for animal and human use), and in particular those reagents and methods suitable for formulating protein pharmaceuticals for animal, and particularly human, use.

[0235] Accordingly, many methods and components well known and routine in the relevant art for the formulation and use of pharmaceutical agents can be used in the design, manufacture and use of self-buffering protein formulations in accordance with the various aspects and preferred embodiments of the invention related thereto. Such methods and components are described in REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21st ed.; Beringer et al., eds., Lippincott, Williams & Wilkins, Philadelphia, PA (2005); ANSEL'S PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVARY SYSTEMS, 8th ed., Allen et al., eds., Lippincott, Williams & Wilkins, Philadelphia, PA (2005); and PHARMACEUTICAL FORMULATION OF PEPTIDES AND PROTEINS, Sven Frokjaer and Lars Hovgaard, eds., CRC Press, Boca Raton, Florida (2000), to name a few readily available references in this regard, each of which is incorporated herein in its entirety, particularly in the portions relating to conventional components and methods that may be used in self-buffering formulations of proteins according to various aspects and preferred embodiments of the present invention related thereto.

[0236] Further methods and compositions that may be useful in this regard are described in, inter alia, U.S. Pat. No. 6,171,586; International Publication No. WO 2005 / 044854; U.S. Pat. No. 6,288,030; U.S. Pat. No. 6,267,958; International Publication No. WO 2004 / 055164; U.S. Pat. No. 4,597,966; U.S. Pat. No. 2003 / 0138417; U.S. Pat. No. 6,252,055; U.S. Pat. No. 5,608,038; U.S. Pat. No. 6,875,432; U.S. Application Publication No. 2004 / 0197324; International Publication No. WO No. 5,908,826; European Patent No. 0 804 163; and International Publication No. WO 2005 / 063291, each of which is incorporated herein by reference in its entirety, particularly in the portions relating to pharma- ceutical acceptable self-buffering protein formulations according to the present invention.

[0237] Various specific aspects of the components and specific types of formulations are further described below by way of example, and the description thus provided is not intended to be exhaustive or in any way exclusive of the methods and compositions applicable to self-buffering protein formulations according to various aspects and embodiments of the invention.

[0238] In preferred embodiments of the various aspects of the invention, the formulation of the self-buffering protein comprises the protein and a carrier. The carrier may be variously referred to herein as one or more of a vehicle, a primary vehicle, a diluent, a primary diluent, a primary carrier, a solvent and / or a primary solvent, depending on the case. In the broadest sense, the carrier may be a gas, a liquid or a solid, as appropriate for the composition and / or the mode of its use. In some embodiments of the invention in this regard, the carrier is a solid, such as a powder, in which the protein can be dispersed. In preferred embodiments in this regard, the carrier is a liquid, particularly one in which the self-buffering protein is highly soluble at a concentration that provides the desired buffering capacity. The liquid carrier may be an organic or non-organic carrier. Preferably, these liquid carriers are aqueous, and most preferably, they are composed primarily or entirely of pure water.

[0239] It will be understood that formulations for pharmaceutical use according to various aspects and embodiments of the invention must be compatible with the methods and conditions to which they will be exposed, such as sterilization (usually applied prior to mixing with the active agent) and conditions during storage.

[0240] In most cases, formulations according to many aspects and embodiments of the invention will include additional components such as excipients and other pharmaceutical agents, but are not limited in any way to this, and in any event, it should be understood that the formulations according to the invention are self-buffering formulations, the buffering capacity of which is provided substantially or entirely by the primary protein itself, as described elsewhere herein.

[0241] Formulations according to various aspects and embodiments of the invention may include, inter alia, the following excipients, including, but not limited to, ingredients to modify, maintain or preserve, e.g., osmolality, osmolarity, viscosity, clarity, color, tonicity, odor, sterility, stability, rate of dissolution or release, rate of adsorption or penetration of the formulation and / or the subject polypeptide and / or protein.

[0242] Of course, the formulation will depend on, among other things, the particular protein being formulated, other active agents, such as other pharmaceutical agents, included in the formulation, the intended route of administration, the method of administration utilized, the amount administered, the frequency of administration, and the form of delivery.

[0243] Formulations according to certain preferred embodiments of various aspects of the invention provide compositions comprising a protein, preferably a pharmaceutical protein, and a solvent, wherein the protein has a buffering capacity of at least about: 2.0 or 3.0 or 4.0 or 5.0 or 6.50 or 8.00 or 10.0 or 15.0 or 20.0 or 30.0 or 40.0 or 50.0 or 75.0 or 100 or 125 or 150 or 200 or 250 or 300 or 350 or 400 or 500 or 700 or 1,000 or 1,500 or 2,000 or 2,500 or 3,000 or 4,000 or 5,000 mM sodium acetate buffer over the pH range of 5.0-4.0 or 5.0-5.5, measured as described in Example 1 or 2 and elsewhere herein.

[0244] Formulations according to certain preferred embodiments of the various aspects of the invention provide self-buffering protein compositions, particularly pharmaceutical protein compositions, where the buffering capacity per unit volume of the composition, excluding the buffering capacity of the protein, is equal to or less than the buffering capacity of 1.0 or 1.5 or 2.0 or 3.0 or 4.0 or 5.0 mM sodium acetate buffer over the pH range 5.0-4.0 or pH 5.0-5.5, measured as described in Example 1 or 2 and elsewhere herein.

[0245] Formulations according to certain preferred embodiments of the various aspects of the invention provide self-buffering protein compositions, particularly pharmaceutical protein compositions comprising a protein and a solvent, where at a pH of the composition the buffering capacity of the protein is at least about: 1.00 or 1.50 or 1.63 or 2.00 or 3.00 or 4.00 or 5.00 or 6.50 or 8.00 or 10.0 or 15.0 or 20.0 or 30.0 or 40.0 or 50.0 or 75.0 or 100 or 125 or 150 or 200 or 250 or 300 or 350 or 400 or 500 or 700 or 1,000 or 1,500 or 2,000 or 2,500 or 3,000 or 4,000 or 5,000 mEq / l / 1 pH unit of pH change.

[0246] Certain preferred embodiment formulations of various aspects of the invention provide self-buffering protein compositions, particularly pharmaceutical protein compositions comprising a protein and a solvent, wherein the buffering capacity per unit volume of the composition, excluding the protein, at a pH of the composition is equal to or less than the buffering capacity of 0.50 or 1.00 or 1.50 or 2.00 or 3.00 or 4.00 or 5.00 or 6.50 or 8.00 or 10.0 or 20.0 or 25.0 mM acetate buffer over the pH range 5.0-4.0 or pH 5.0-5.5, measured as described in Example 1 or 2 and elsewhere herein.

[0247] Certain preferred embodiment formulations of various aspects of the invention provide self-buffering protein compositions, particularly pharmaceutical protein compositions comprising a protein and a solvent, where at a desired pH, the protein provides a buffering capacity of at least about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of the buffering capacity of the composition.

[0248] Certain preferred embodiment formulations of various aspects of the present invention provide self-buffering protein compositions, particularly pharmaceutical protein compositions, comprising a protein and a solvent, wherein the protein has a concentration of about: 20-400, or 20-300, or 20-250, or 20-200, or 20-150 mg / ml.

[0249] Certain preferred embodiment formulations of various aspects of the invention provide self-buffering protein compositions, particularly pharmaceutical protein compositions comprising a protein and a solvent, wherein the pH maintained by the buffering action of the protein is about: pH 3.5-8.0, or 4.0-6.0, or 4.0-5.5, or 4.5-5.5.

[0250] Formulations according to certain preferred embodiments of the various aspects of the present invention provide self-buffering protein compositions, particularly pharmaceutical protein compositions comprising a protein and a solvent, wherein the salt concentration of the composition is less than: 150 mM, or 125 mM, or 100 mM, or 75 mM, or 50 mM, or 25 mM.

[0251] Certain preferred embodiment formulations of various aspects of the present invention provide self-buffering protein compositions, particularly pharmaceutical protein compositions, comprising a protein and a solvent, and further comprising one or more of: a pharma- ceutically acceptable salt; an osmotic balancing agent (tonicity agent); an antioxidant; an antibiotic; an antifungal agent; a bulking agent; a lyoprotectant; an antifoaming agent; a chelating agent; a preservative; a coloring agent; an analgesic agent; or an additional drug.

[0252] Formulations according to certain preferred embodiments of the various aspects of the present invention provide self-buffering protein compositions, in particular pharmaceutical protein compositions, comprising a protein and a solvent and further comprising a hypotonic, isotonic or hypertonic, preferably near isotonic, particularly preferably an isotonic amount of one or more pharma- ceutically acceptable polyols, very preferably any one or more of sorbitol, mannitol, sucrose, trehalose or glycerol, particularly very preferably about 5% sorbitol, 5% mannitol, 9% sucrose, 9% trehalose or 2.5% glycerol, in particular 5% sorbitol, 5% mannitol, 9% sucrose, 9% trehalose or 2.5% glycerol in this regard.

[0253] Certain preferred embodiment formulations of the various aspects of the invention provide self-buffering protein compositions, particularly pharmaceutical protein compositions, comprising a protein and a solvent and further comprising one or more pharma- ceutical acceptable surfactants, preferably one or more of polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan, polyethoxylates, and poloxamer 188, particularly preferably polysorbate 20 or polysorbate 80, preferably about 0.001-0.1% polysorbate 20 or polysorbate 80, most preferably about 0.002-0.02% polysorbate 20 or polysorbate 80, particularly preferably 0.002-0.02% polysorbate 20 or polysorbate 80.

[0254] Formulations according to certain preferred embodiments of various aspects of the invention provide self-buffering protein compositions, particularly pharmaceutical protein compositions comprising a protein and a solvent, wherein the protein is a pharmaceutical product, and the composition is a sterile formulation of the protein suitable for treatment of an animal or human medical subject.

[0255] Formulations according to various aspects and embodiments of the invention described herein are in particular lyophilized compositions as described above that upon reconstitution provide the formulations described above and elsewhere herein.

[0256] a. Excipients and other additional ingredients As mentioned above, certain embodiments according to this aspect of the invention provide self-buffering protein compositions, particularly pharmaceutical protein compositions, which in addition to a protein, particularly a pharmaceutical protein, comprise one or more excipients as specifically described in this section and elsewhere herein. In this regard, excipients are used in the present invention for a wide range of purposes, such as adjusting the physical, chemical or biological properties of the formulation, adjusting the viscosity, and / or the processes of the present invention to improve the efficacy and / or stabilize such formulations, as well as against deterioration and damage due to stresses arising during and after manufacture, transportation, storage, processing prior to use, administration.

[0257] Various reviews of protein stabilization and formulation, materials and methods useful in this regard, are available, e.g., Arakawa et al., "Solvent interactions in pharmaceutical formulations," Pharm Res. 8(3): 285-91 (1991); Kendrick et al., "Physical stabilization of proteins in aqueous solution," in RATIONAL DESIGN OF STABLE PROTEIN FORMULATIONS: THEORY AND PRACTICE, Carpenter and Manning, eds. Pharmaceutical Biotechnology. 13: 61-84 (2002), and Randolph et al., "Surfactant-protein interactions," Pharm Biotechnol. 13: 159-75 (2002), each of which is incorporated by reference in its entirety, particularly in the portions relating to excipients and processing thereof for self-buffering protein formulations according to the present invention, and particularly with respect to protein pharmaceutical products and methods for veterinary and / or human medical use.

[0258] Various excipients useful in the present invention are listed in Table 1 and further described below. [Table 1]

[0259] i. salt Salts may be used in accordance with certain preferred embodiments of the invention, for example, to adjust the ionic strength and / or tonicity of the self-buffering formulation and / or to improve the solubility and / or physical stability of the self-buffering protein or other components of the self-buffering protein composition according to the invention.

[0260] As is well known, ions can stabilize the native state of proteins by binding to charged residues on the protein surface and by shielding charged and polar groups in proteins, reducing the strength of their electrostatic interactions, attractions and repulsions. Ions can also stabilize the denatured state of proteins, particularly by binding to the denatured peptide bonds (-CONH) of the protein. Furthermore, ionic interactions of charged and polar groups in a protein can also reduce intermolecular electrostatic interactions, thereby preventing or reducing protein aggregation and insolubilization.

[0261] Ionic species vary significantly in their effects on proteins. A large number of categorical permutations of ions and their effects on proteins have been described and can be used to formulate self-buffering protein compositions according to the present invention. One example is the Hofmeister series, which permutes ionic and polar nonionic solutes according to their effect on the conformational stability of proteins in solution. Stabilizing solutes are called "kosmotropic". Destabilizing solutes are called "chaotropic". Cosmotropes are typically used at high concentrations (e.g., greater than 1 molar ammonium sulfate) to precipitate proteins out of solution ("salting-out"). Chaotropes are typically used to denature and / or stabilize proteins ("salting-in"). The relative effect of an ion on "salting-in" and "salting-out" defines the ion's position in the Hofmeister series.

[0262] In addition to their (above) usefulness and drawbacks, salts are also effective in reducing the viscosity of protein formulations and may be used in the present invention for this purpose.

[0263] In order to maintain isotonicity in parenteral formulations according to preferred embodiments of the invention, improve protein solubility and / or stability, improve viscosity characteristics, avoid deleterious salt effects on protein stability and aggregation, and prevent salt-mediated proteolysis, the salt concentration in self-buffered formulations according to various preferred embodiments of the invention is less than 150 mM (for monovalent ions) and less than 150 mEq / L for multivalent ions. In this regard, in certain particularly preferred embodiments of the invention, the total salt concentration is from about 75 mEq / L to about 140 mEq / L.

[0264] ii. amino acid Free amino acids can be used in the protein formulations according to various preferred embodiments of the present invention as bulking agents, stabilizers and antioxidants, to name a few.However, the amino acids contained in the self-buffering protein formulations according to the present invention do not provide buffering effects.For this reason, amino acids with significant buffering capacity are not used, and are not used at any pH before or after the pH at which these amino acids have significant buffering activity, or are used at low concentrations, and as a result, the buffering capacity of the amino acids in the formulation is not significant.This is especially true for histidine and other amino acids that are commonly used as buffers in pharmaceutical formulations.

[0265] Subject to the above considerations, lysine, proline, serine and alanine can be used to stabilize proteins in the formulation. Glycine is useful to ensure the correct cake structure and properties in lyophilisates. As a result, glycine is a common component in lyophilized formulations and reconstituted lyophilisates such as Neumega®, Genotropin®, and Humatrope®. Arginine can be useful to inhibit protein aggregation in both liquid and lyophilized formulations such as Activase®, Avonex®, and Enbrel® liquid. Methionine is useful as an antioxidant.

[0266] iii. Polyol Polyols include sugars (e.g., mannitol, sucrose, and sorbitol) and polyhydric alcohols (e.g., glycerol and propylene glycol), and, for the purposes of discussion herein, polyethylene glycol (PEG) and related substances. Polyols are cosmotropic (have a strong affinity for water). They are useful stabilizers to protect proteins from physical and chemical degradation processes in both liquid and lyophilized formulations. Polyols also aid in adjusting the tonicity of the formulation.

[0267] In this regard, one of the polyols useful in the present invention is mannitol, which is commonly used to ensure the structural stability of the cake in lyophilized formulations, such as Leukine®, Enbrel®-Lyo, and Betaseron®. Mannitol ensures the structural stability of the cake. Mannitol is usually used together with a lyoprotectant, such as sucrose. Sorbitol and sucrose are preferred agents for adjusting tonicity and also serve as stabilizers to protect against freeze-thaw stress during transportation or bulk generation during the manufacturing process. Reducing sugars (containing free aldehyde or ketone groups), such as glucose and lactose, can glycate surface lysine and arginine residues. Thus, glucose and lactose are usually not preferred polyols for use according to the present invention. Furthermore, sugars that form reactive species, such as sucrose, which is hydrolyzed under acidic conditions to fructose and glucose, resulting in glycation, are also not preferred amino acids of the present invention in this regard. PEG is useful for protein stabilization and as a cryoprotectant and in this regard can be used in the present invention, such as in Recombinate®.

[0268] iv. Surfactants Protein molecules are susceptible to surface adsorption and denaturation and resulting aggregation at gas-liquid, solid-liquid, and liquid-liquid interfaces. These effects are usually inversely proportional to the protein concentration. These deleterious interactions are usually inversely proportional to the protein concentration and are typically exacerbated by physical agitation that occurs during product transportation and handling.

[0269] Surfactants are routinely used to prevent, minimize, or reduce surface adsorption. Surfactants useful in the present invention in this regard include polysorbate 20, polysorbate 80, polyethoxylates, which are other fatty acid esters of sorbitan, and poloxamer 188.

[0270] Detergents are also commonly used to control the conformational stability of proteins, and their use in this regard is protein specific, as any given detergent will typically stabilize some proteins and destabilize others.

[0271] Polysorbates are susceptible to oxidative degradation and, as supplied, often contain sufficient amounts of peroxide to cause oxidation of the side chains of protein residues, especially methionine. For this reason, polysorbates must be used with caution and, if used, at their minimum effective concentrations. In this regard, polysorbates are a good example of the principle that excipients should be used at their minimum effective concentrations.

[0272] v. antioxidants Various methods can result in the harmful oxidation of proteins in pharmaceutical formulations. The harmful oxidation of proteins can be prevented to some extent by maintaining the proper levels of ambient oxygen and temperature in pharmaceutical formulations and avoiding exposure to light. Antioxidant excipients can also be used to prevent oxidative degradation of proteins. Useful antioxidants in this regard are reducing agents, oxygen / free radical scavengers, and chelating agents. Antioxidants for therapeutic protein formulations according to the present invention are preferably water-soluble and maintain the activity of the protein throughout the shelf life of the product. EDTA is the preferred antioxidant of the present invention in this regard and can be used in the present invention in much the same way as it has been used in formulations of acidic fibroblast growth factor and products such as Kineret® and Ontak®.

[0273] Antioxidants can damage proteins. For example, reducing agents, such as glutathione in particular, can break intramolecular disulfide bonds. Thus, the antioxidants for use in the present invention are specifically selected to eliminate or substantially reduce the possibility that they themselves damage the proteins in the formulation.

[0274] vi. Metal ions The formulations according to the invention may contain metal ions that are cofactors for proteins and are necessary to form protein coordination complexes (e.g., zinc, which is necessary to form certain insulin suspensions). Metal ions may also inhibit some processes that break down proteins. However, metal ions also catalyze the physical and chemical processes that break down proteins.

[0275] Magnesium ions (10-120 mM) can also inhibit the isomerization of aspartic acid to isoaspartic acid. +2 ions (up to 100 mM) can increase the stability of human deoxyribonuclease (rhDNase, Pulmozyme®). However, Mg +2 , Mn +2 , and Zn +2 may destabilize rhDNase. +2 and Sr +2 can stabilize factor VIII, but this factor is +2 , Mn +2 and Zn +2 , Cu +2 and Fe +2 The aggregation of this factor can be destabilized by Al. +3 It can be increased by ions.

[0276] vii. Preservatives Preservatives are necessary when developing multi-dose parenteral formulations with more than one withdrawal from the same container. The main function of a preservative is to prevent microbial growth and ensure sterility of the product throughout the shelf life or use of the drug. Commonly used preservatives include benzyl alcohol, phenol, and m-cresol. Although preservatives have a long history of use with small molecule parenterals, developing protein formulations containing preservatives is difficult. Preservatives almost always have a destabilizing effect on proteins (aggregation), which is the main factor limiting their use in multi-dose protein formulations. To date, most protein drugs have been formulated for single use only. However, a multi-dose formulation would have the added benefit of being more convenient for patients and more marketable. A good example is human growth hormone (hGH), the development of which has led to the commercialization of the introduction of more convenient, multi-use injection pens. At least four such pen devices containing preserved formulations of hGH are currently on the market. Norditropin® (liquid, Novo Nordisk), Nutropin AQ® (liquid, Genentech) & Genotropin (lyophilized-dual chamber cartridge, Pharmacia & Upjohn) contain phenol, while Somatrope® (Eli Lilly) is formulated with m-cresol.

[0277] Several aspects need to be considered during the formulation and development of preserved formulations. The effective preservative concentration in the pharmaceutical product must be optimized. This fact necessitates testing a given preservative in said formulation with a concentration range that provides antimicrobial efficacy without compromising protein stability. For example, in the development of a liquid formulation for Interleukin-1 receptor (type I), three preservatives were successfully screened using Differential Scanning Calorimetry (DSC). These preservatives were ranked based on their impact on stability at concentrations commonly used in commercial products.

[0278] As expected, developing liquid formulations containing preservatives is more challenging than lyophilized formulations. Lyophilized products can be lyophilized without preservatives and reconstituted with a preservative-containing diluent at the time of use. This reduces the time the preservative is in contact with the protein, significantly minimizing stability risks associated with contact. When used in liquid formulations, the efficacy and stability of the preservative must be maintained over the entire shelf life of the product (~18-24 months). An important point to keep in mind is that the efficacy of the preservative must be demonstrated in the final formulation containing the active drug and all excipient components.

[0279] Self-buffering protein formulations, in particular self-buffering biopharmaceutical protein formulations, according to the present invention will usually be designed with a particular range of bioavailability and duration, specifically for a particular route and method of administration, a particular dosage and frequency of administration, a particular treatment and a particular disease.

[0280] Thus, formulations in accordance with the present invention can be designed for delivery by any suitable route, including, but not limited to, oral, auricular, opthalmically, rectally, and vaginally, as well as parenteral routes, such as intravenous and intraarterial, intramuscular, and subcutaneous injection.

[0281] b. Parenteral Formulations Preparations for parenteral administration may be in the form of aqueous or non-aqueous isotonic sterile injection solutions or suspensions, which solutions and suspensions may be prepared from sterile powders or granules with one or more of the carriers or diluents mentioned above for use in preparations for oral administration, or with other suitable dispersing or wetting agents and suspending agents.

[0282] When intended for parenteral administration, the therapeutic compositions for use in the present invention may be in the form of a parenterally acceptable aqueous solution containing the desired protein in a pyrogen-free, pharma- ceutically acceptable vehicle. A particularly suitable vehicle for parenteral injection is sterile pure water, in which the protein of the invention is formulated as a sterile, isotonic, self-buffered solution.

[0283] Such formulations may also include the desired protein formulations, particularly in the form of injectable microspheres, bioerodible particles, polymeric compounds (polylactic acid, polyglycolic acid), beads, or liposomes, for example in a form that provides controlled or sustained release. Such formulations may be introduced, particularly by implantable drug delivery devices.

[0284] Formulations for parenteral administration may include substances that modify viscosity, such as carboxymethylcellulose, sorbitol, and dextran. The formulation may also include components that increase the solubility of the desired protein or other components and one or more such components, such as, in some cases, components that stabilize the self-buffering proteins of the invention.

[0285] c. Pulmonary administration formulations The pharmaceutical composition according to certain embodiments of the present invention may be suitable for inhalation. For pulmonary administration, the pharmaceutical composition of the present invention can be administered in aerosol form or by using an inhaler with dry powder aerosol. For example, the binder can be formulated as a dry powder for inhalation. The inhalation solution can also be formulated with a propellant for aerosol delivery. In yet another embodiment, the solution can be nebulized. Pulmonary administration is further described in International Patent Application No. PCT / US94 / 001875, which describes pulmonary delivery of chemically modified proteins.

[0286] d. Oral Formulations For oral administration, the pharmaceutical composition of the present invention can be in the form of, for example, a tablet, capsule, suspension, or liquid. The pharmaceutical composition of the present invention is preferably prepared in the form of a dosage unit containing a specific amount of the active ingredient. Examples of such dosage units are tablets or capsules. In this regard, the formulations for oral administration according to the present invention can be prepared in a conventional manner, where buffering in the formulation is provided by the self-buffering proteins described elsewhere herein.

[0287] e. Controlled-release formulations Additional formulations described herein that may be useful in the present invention include sustained and controlled delivery formulations. Techniques for producing such sustained and controlled delivery formulations that may be used in accordance with various aspects and preferred embodiments of the present invention are well known to those skilled in the art. These techniques include delivery methods using liposome carriers, bioerodible microparticles, porous beads, and semipermeable polymer matrices, e.g., International Patent Application No. PCT / US93 / 00829; U.S. Pat. No. 3,773,919; European Patent No. 58,481; Sidman et al., Biopolymers, 22:547-556 (1983); Langer et al., J. Biomed. Mater. Res., 15:167-277, (1981); Langer et al., Chem. Tech., 12:98-105 (1982); European Patent No. 133,988; Eppstein et al., Proc. Natl. Acad. Sci. (USA), 82:3688-3692. (1985); European Patent No. 36,676; European Patent No. 88,046; and European Patent No. 143,949, each of which is incorporated by reference in its entirety herein, particularly in those portions relating to the self-buffering sustained- and controlled-delivery pharmaceutical protein formulations of the present invention described herein.

[0288] f. sterile The pharmaceutical composition of the present invention used for in vivo administration must usually be sterile. Sterilization is achieved by filtration through a sterile filtration membrane. If the composition is lyophilized, sterilization using this method can be performed either before or after lyophilization and reconstitution. The composition for parenteral administration can be stored in lyophilized form or in a solution. In addition, parenteral compositions are usually placed in a container with a sterile access port, for example, an intravenous solution bag or vial with a stopper that can be pierced by a hypodermic injection needle.

[0289] g. keep Once formulated, the pharmaceutical compositions of the invention may be stored in sterile vials as solutions, suspensions, gels, emulsions, solids, or dehydrated or lyophilized powders. Such formulations may be stored either in a ready-to-use form or in a form requiring reconstitution prior to administration (e.g., lyophilized).

[0290] h. Additional Agents The self-buffering protein composition, particularly the self-buffering pharmaceutical protein composition, according to the present invention may contain one or more additional agents in addition to the self-buffering protein of the composition. Such agents may be proteins or other types of agents. Such agents include agents for the prevention or treatment of any disorder or disease. Such agents include, for example, antibiotics and antifungals. Additional agents also include agents for the treatment of human disorders, including, but not limited to, agents for the treatment of inflammatory diseases, cancer, metabolic disorders, neurological disorders, and renal disorders, to name a few. In this regard, agents that can be used in the present invention also include agents that are useful for enhancing the action of the self-buffering compositions and / or preventing, ameliorating, or treating any undesirable side effects of their administration.

[0291] i. Method for producing self-buffering protein formulations The compositions according to the present invention can be prepared using well-known and routine methods for preparing, formulating and using proteins, particularly pharmaceutical proteins. In certain preferred embodiments of many aspects of the present invention, the method for preparing the compositions of the present invention includes the use of counterions to remove residual buffers. In this regard, the term counterion refers to any polar or charged component that serves to displace buffers from the composition during preparation of the composition. Useful counterions in this regard include, for example, glycine, chloride, sulfate and phosphate. In this regard, the term counterion is used to mean roughly the same thing as displacing ion.

[0292] Residual buffer can be removed using this counterion using a variety of well-known methods, including, but not limited to, standard methods of dialysis and high performance membrane diffusion-based methods such as cross-flow diafiltration. In this regard, methods for residual buffer removal using counterions can also optionally be carried out using size exclusion chromatography.

[0293] In certain related preferred embodiments in this regard, the compositions according to the invention are prepared by a method involving dialysis against an unbuffered solution having a pH lower than the pH of the formulation containing the self-buffering protein. In particularly preferred embodiments of the invention in this regard, said unbuffered solution comprises a counterion, in particular a counterion that facilitates removal of residual buffer and that does not adversely affect the self-buffering protein or its formulation. In a further particularly preferred embodiment of the invention, after dialysis, the pH of the formulation is adjusted to the desired pH using dilute acid or dilute base.

[0294] In certain related preferred embodiments in this regard, the compositions according to the invention are prepared by a process involving cross-flow diafiltration against an unbuffered solution having a pH lower than the pH of the formulation containing the self-buffering protein. In particularly preferred embodiments of the invention in this regard, said unbuffered solution comprises a counterion, in particular a counterion that facilitates the removal of residual buffer and that does not adversely affect the self-buffering protein or its formulation. In a further particularly preferred embodiment of the invention, after diafiltration, the pH of the formulation is adjusted to the desired pH using dilute acid or dilute base.

[0295] 5. Route of administration In various embodiments, the formulations according to the present invention can be administered by various suitable routes well known to those skilled in the art of administering therapeutic agents to subjects.In an embodiment of the present invention in this regard, one or more formulations are administered via the digestive tract, as described elsewhere herein.In other embodiments, one or more formulations are administered parenterally, as described elsewhere herein.In various embodiments, one or more formulations can be administered via the digestive tract in combination with one or more other formulations that are administered parenterally.

[0296] Such routes in various embodiments include, but are not limited to, oral, ocular, mucosal, topical, rectal, pulmonary (e.g., by inhalation spray), and transdermal administration of the compositions of the invention. The following parenteral routes of administration are also useful in various embodiments of the invention: intravenous, intraarterial, intracardiac, intraspinal, intrathecal, intraosseous, intraarticular, intrasynovial, intradermal, subcutaneous, peritoneal, and / or by intramuscular injection.

[0297] In some embodiments, intravenous, intraarterial, intradermal, subcutaneous and / or intramuscular injections are used. In some embodiments, intravenous, intraarterial, intradermal, subcutaneous and / or intramuscular injections are used.

[0298] In certain embodiments of the invention, the compositions are administered locally, for example, by intraocular injection to treat ocular neovascularization, retinopathy, or age-related macular degeneration.

[0299] 6. Dosage The amount of self-buffering protein formulation to be administered and the administration regimen for treating disease state with this formulation depend on various factors, such as the age, weight, sex and condition of the subject, the type of disease, the severity of disease, the route and frequency of administration, and the specific formulation to be used.In particular, the amount depends on the protein therapeutic agent to be administered and any other therapeutic agent to be administered in combination therewith.The dosage can be determined for the formulation according to the present invention using the conventional pharmaceutical method well established for this purpose.

[0300] 7. Dosing regimen The formulations of the present invention can be administered in dosages and by techniques well known to those of ordinary skill in the medical and veterinary arts, taking into account factors such as the age, sex, weight, and condition of the particular patient, as well as the formulation to be administered (e.g., solid formulation vs. liquid formulation). Dosages for humans or other mammals can be determined from the disclosures herein, the literature cited herein, and knowledge in the art without undue experimentation by those of ordinary skill in the art.

[0301] According to various embodiments, the appropriate dosage and administration schedule will depend on many factors and may vary under different circumstances. The parameters that determine the optimal dosage schedule of administration will typically include some or all of the following: the disease being treated and its stage; the species of the subject, its health condition, sex, age, weight, and metabolic rate; other therapeutic agents being administered; and potential complications expected from the medical history or genotype of the subject.

[0302] The optimal dosing regimen for a given situation will also take into account the nature of the formulation, the method of administration, the route of distribution after administration, and the rate of elimination both from the site of action and from the subject's body. Ultimately, the determination of optimal dosing will preferably provide an effective dose that is neither below a threshold of maximum efficacy nor above a threshold at which adverse effects associated with doses of active agent outweigh the benefits of increasing doses.

[0303] It will be understood that a "dose" may be delivered all at once, in divided doses, or continuously over an extended period of time. The entire dose may be delivered to a single site, or may be partially spread over several sites in divided doses. Furthermore, the dose may remain the same during treatment or may vary.

[0304] In various embodiments, the formulations of the invention are administered at an initial dosage, followed by additional administrations to maintain that amount. In some embodiments, the formulations of the invention are administered initially by one method, followed by the same or one or more different methods. The dosage of the ongoing administration can be adjusted to maintain the level of the active agent in the subject at a particular value. In some embodiments, the compositions of the invention are administered initially and / or by intravenous injection to maintain the level of the composition in the subject. In various embodiments, other forms of administration are used.

[0305] The formulations of the present invention can be administered at many frequencies over a wide range of times, for example, at any suitable frequency and time range that delivers a therapeutically effective amount. Doses can be delivered and administered every few hours, once or more per day, every day, every other day, or several times per week, or less frequently and continuously. In some embodiments, the formulations of the present invention are administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more. In some embodiments, the formulations of the present invention are administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more. In various embodiments, the formulations of the present invention are administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more. In various embodiments, the formulations of the present invention are administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 years or more. The suitable dosage schedule of the initial dose and the additional dose for the continuous administration may be all the same dosage amount or may vary.The suitable dosage schedule can be determined by those skilled in the art from the disclosure of this specification, the references cited in this specification, and the common general knowledge.Generally, the duration of treatment will be proportional to the duration of disease progression, the effectiveness of the treatment applied, and the condition and response of the subject to be treated.

[0306] 8. Disease and Treatment In a preferred embodiment, the self-buffering pharmaceutical protein composition according to the present invention is useful for treating a number of subjects suffering from a variety of diseases and disorders. As mentioned elsewhere herein, the present invention provides in particular self-buffering compositions of pharmaceutical antibodies, antibody-derived pharmaceutical proteins, and antibody-related pharmaceutical proteins that may contain Fc effector functions and binding domains specific for a wide range of disease-related targets and are useful for treating diseases. These proteins and their self-buffering compositions are described in detail herein above, and their use in treating various disorders and diseases related to these targets is also described. Methods of using the compositions of the present invention, including methods of formulation, methods of administration, dosages, and methods of administration, are all specifically described above. The formulation and administration of any particular composition of the present invention can be tailored to treat a particular disease using routine techniques well known in the art, taking into account the guidance provided by the description of the present invention. Diseases that are effectively treated using self-buffering pharmaceutical protein formulations according to various aspects and preferred embodiments of the present invention include inflammatory diseases, cancer, metabolic disorders, neurological disorders, and renal disorders, to name a few.

[0307] 9. Packaging and Kits The invention also provides kits comprising self-buffering protein formulations, particularly kits comprising in one or more containers a self-buffering pharmaceutical protein formulation and instructions for its use, particularly where the formulation is a pharma- ceutical formulation for human use. Preferred kits include those comprising in one or more containers a self-buffering protein formulation of the invention and one or more separate documents, information regarding the contents of the kit and / or its use, particularly where the protein is a biopharmaceutical protein formulated for the treatment of a human disease.

[0308] In certain aspects of the invention in this regard, preferred kits include those kits described above which further comprise one or more single-chambered or multi-chambered syringes (e.g., liquid syringes and lyosyringes) for administering one or more self-buffering protein formulations of the invention. In certain aspects of the invention in this regard, certain particularly preferred kits further comprise preloaded syringes. In even more particularly preferred embodiments in this regard, the above kits comprise a self-buffering pharmaceutical composition for parenteral administration sealed in a vial under partial vacuum, ready to be loaded into a syringe and administered to a subject. In a particularly preferred embodiment, the composition of the invention is placed in a vial under partial vacuum. In this and all other respects, in certain more particularly preferred embodiments, the kit of the invention comprises one or more vials according to any of the above, wherein each vial comprises a single dose to be administered to a subject. In all these and other respects, the invention further relates to a kit comprising said lyophilisate, placed as described above, which upon reconstitution provides the composition according to the lyophilisate. In this regard, the invention further provides in certain preferred embodiments thereof a kit comprising a lyophilisate according to the invention and a sterile diluent for reconstituting said lyophilisate. EXAMPLES

[0309] The invention is further described by the following illustrative, non-limiting examples.

[0310] Example 1: Acid titration of sodium acetate buffer and buffer capacity in the pH range 5.0-4.0 Stock solutions of acetate of known concentrations were prepared by diluting ultrapure glacial acetate in HPLC grade water and then titrating the pH to the desired value with NaOH. The stock solutions were equilibrated with air and 21° C. Volumetric standards were prepared at a concentration of 1 N and diluted as necessary with HPLC water.

[0311] Sodium acetate buffers of 1 mM, 2.5 mM, 5 mM, 7.5 mM, 10 mM, and 15 mM were prepared by diluting the stock solution in HPLC water. The solutions were titrated with HCl. 0.2 N HCl was used for the 1, 2.5, and 5 mM solutions, 0.4 N HCl was used for the 7.5 mM solution, and 0.8 N HCl was used for the 10 and 15 mM solutions. The titrations were performed using standard analytical laboratory techniques.

[0312] Panel A of Figure 1 shows the titration data and least squares fit curves calculated from the data for each solution. The slope of the curve calculated from each data set was taken as the buffer capacity of the corresponding acetate buffer. The linear dependence of buffer capacity on acetate buffer concentration is shown in Panel B of Figure 1.

[0313] Example 2: Base titration of sodium acetate buffer and buffer capacity in the pH range 5.0-5.5 Acetate buffer stock and titration solutions were prepared as described in Example 1. The titration solutions were titrated as described in Example 1, except that they were titrated to pH 5.0-5.5 and were titrated with NaOH instead of HCl. 0.2 N NaOH was used to titrate the 1, 2.5, and 5 mM solutions, and 0.4 N NaOH was used for the 7.5, 10, and 15 mM solutions. The titration results are shown in Figure 2A. The linear dependence of buffer capacity on acetate buffer concentration is shown in Figure 2B.

[0314] Example 3: Measurement of Acetate by HPLC Acetate was measured in acetate buffer samples using analytical SE-HPLC. A standard curve of peak area as a function of acetate concentration was established by analysis of acetate in buffers of known acetate concentrations. The amount of acetate in the test samples was interpolated from this standard curve. The standard curve is shown in Figure 3. The nominal and measured amounts of acetate in the test buffers are tabulated below the standard curve in the figure.

[0315] Example 4: Acid titration of Ab-hOPGL formulations over the range pH 5.0 to pH 4.0 Bulk Ab-hOPGL in 10 mM acetate (nominal), 5% sorbitol, pH 5.0 was diafiltered against 5.25% sorbitol, pH 3.2 (adjusted with HCl) in a LABSCALE TFF® system (Millipore Corp.) using 3 Millipore Pellicon XL 50 regenerated cellulose ultrafiltration membranes using a multi-manifold cassette. The diafiltrate was exchanged 8-10 times during the diafiltration of each formulation. After diafiltration, the pH of the resulting unbuffered solution was measured and adjusted to pH 5.0 using 0.05 N HCl or 0.05 N NaOH.

[0316] Solutions of 1, 10, 30, 60, 90, and 110 mg / ml were prepared for titration by dilution. The pH of each dilution was adjusted to pH 5.0 with NaOH or HCl, as necessary. Titrations were performed as described in the examples above. 0.2 N HCl was used to titrate the 1, 10, and 30 mg / ml solutions. 0.4 N HCl was used to titrate the 60 mg / ml solutions. 0.8 N HCl was used to titrate the 90 and 110 mg / ml solutions.

[0317] The titration results are shown in Figure 4. Least squares regression lines are shown for each concentration data set. Buffer capacity is taken as the slope of the regression line for each concentration.

[0318] Example 5: Base titration of Ab-hOPGL formulations over the pH range 5.0-6.0 Ab-hOPGL solutions of 1, 10, 30, 60, 90, and 110 mg / ml were prepared for titration as described in Example 4. Base titrations were performed with NaOH as described in the previous example. 0.2 N NaOH was used for the 1, 10, 30, and 60 mg / ml solutions, and 0.4 N NaOH was used for the 90 and 110 mg / ml solutions. The titration results are depicted in the graph of FIG. 5. A linear regression line is shown for the data at each concentration. The buffer capacity is taken as the slope of the regression line for each concentration.

[0319] Example 6: Residual acetate levels in self-buffered Ab-hOPGL formulations The amount of residual acetate in the Ab-hOPGL formulations was measured using the method described in Example 3. The results are presented in Figure 6, which is a graph showing the standard curve for HPLC measurement of acetate concentration, and below the graph is a table of the results of measurements performed on Ab-hOPGL formulations of different concentrations. The Ab-hOPGL concentrations are shown on the left ("nominal measurements") and the measured acetate concentration for each Ab-hOPGL concentration is shown on the right.

[0320] Example 7: Buffer capacity of Ab-hOPGL formulations ± residual acetate in the pH range 5.0-4.0 Self-buffered Ab-hOPGL formulations were prepared and titrated with HCl as described in the examples above. Additionally, the data were adjusted by subtracting the residual acetate buffering contribution based on the measurement of acetate content, for example, by SE-HPLC as described in Example 3. Buffering capacity was measured as described above. The same analysis was performed on both the positive and negative data sets. The results presented in Figure 7 show the impact of residual acetate on the buffering capacity of Ab-hOPGL formulations. The results reveal that the buffering capacity of residual acetate is a minor factor in the buffering capacity of the analyzed self-buffered Ab-hOPGL formulations.

[0321] Example 8: Buffer capacity of Ab-hOPGL ± residual acetate in the pH range 5.0-6.0 Self-buffered Ab-hOPGL formulations were prepared and titrated with NaOH as described in the examples above. Additionally, the data were adjusted by subtracting the residual acetate buffering contribution based on the measurement of acetate content by SE-HPLC as described in Example 3, for example. Buffering capacity was measured as described above. The same analysis was performed on both the positive and negative data sets. The results presented in FIG. 8 show the effect of residual acetate on the buffering capacity of Ab-hOPGL formulations. The results reveal that the buffering capacity of residual acetate is a minor factor in the buffering capacity of the analyzed self-buffered Ab-hOPGL formulations.

[0322] Example 9: pH and Ab-hOPGL stability in self-buffered formulations versus conventional buffers Self-buffered formulations of Ab-hOPGL were prepared as in the examples above. Additionally, formulations were made to contain either the conventional buffering agents acetate or glutamate. All formulations contained 60 mg / ml Ab-hOPGL. The pH in the formulations and the stability of Ab-hOPGL were monitored over a 6 month storage period at 4° C. Stability was monitored by measuring monomeric Ab-hOPGL in the formulations over the storage period. Measurements were performed using SE-HPLC as described above. Results for all three formulations are shown in FIG. 9. Panel A shows the stability of Ab-hOPGL in the three formulations. Stability in the self-buffered formulations is as good as the conventional buffered formulation. Panel B shows the pH stability of the three formulations. Again, pH stability in the self-buffered formulations is as good as the conventional buffered formulation.

[0323] Example 10: Titration and buffer capacity of Ab-hB7RP1 - pH 5.0 to 4.0 Self-buffering formulations of Ab-hB7RP1 were prepared at concentrations of 1, 10, 30, and 60 mg / ml as described for Ab-hOPGL in the above examples. Titrations were performed with HCl as described above. Additionally, the data were adjusted by subtracting the residual acetate buffering contribution based on the measurement of acetate content by SE-HPLC as described in Example 3, for example. Panel A of FIG. 10 shows the titration results. Panel B of FIG. 10 shows the dependence of buffering capacity on Ab-hB7RP1 formulation concentration before and after subtracting the residual acetate buffering contribution. The results clearly demonstrate the self-buffering capacity of Ab-hB7RP1 in this pH range. At 40 mg / ml, the self-buffering formulation of Ab-hB7RP1 provides a buffering capacity in this pH range that is approximately the same as 10 mM sodium acetate buffer. At 60 mg / ml, the formulation provides a buffering capacity that is approximately the same as 15 mM sodium acetate buffer.

[0324] Example 11: Titration and buffer capacity of Ab-hB7RP1 - pH 5.0-6.0 Self-buffering formulations of Ab-hB7RP1 were prepared at concentrations of 1, 10, 30, and 60 mg / ml as described for Ab-hOPGL in the above examples. Titrations were performed with NaOH as described above. Additionally, the data were adjusted by subtracting the residual acetate buffering contribution based on the measurement of acetate content, e.g., by SE-HPLC as described in Example 3. Panel A of FIG. 11 shows the titration results. Panel B of FIG. 11 shows the dependence of buffering capacity on Ab-hB7RP1 formulation concentration, before and after subtracting the residual acetate buffering contribution. The results clearly demonstrate the self-buffering capacity of Ab-hB7RP1 in this pH range. At 60 mg / ml, the self-buffering formulation of Ab-hB7RP1 provides a buffering capacity in this pH range that is approximately the same as 10 mM sodium acetate buffer.

[0325] Example 12: Stability of Ab-hB7RP1 in self-buffered and conventionally buffered formulations at 4°C and 29°C Ab-hB7RP1 was prepared as described in the Examples above and formulated as above in self-buffered formulations and in formulations using either conventional buffers, acetate or glutamate. All formulations contained 60 mg / ml Ab-hB7RP1. Solution pH and stability of Ab-hB7RP1 in solution were monitored over a 26 week storage period at 4° C. or 29° C. Stability was monitored by measuring monomeric Ab-hB7RP1 in the formulations over the storage period. Measurements were performed using SE-HPLC as described above. Results are shown in FIG. 12. Panel A shows results from storage at 4° C. Panel B shows results from storage at 29° C. Ab-hB7RP1 was at least as stable in the self-buffered formulation at 4° C. as the conventional buffer. At 29° C., the self-buffered formulation was at least as stable as the conventional buffer and may have been slightly more stable than the conventional buffer formulation at 10 weeks to end.

[0326] Example 13: pH stability of self-buffered Ab-hB7RP1 at 4°C and 29°C 60 mg / ml of self-buffered Ab-hB7RP1 was prepared as described in the above example. The pH was monitored during storage at the same temperature as described in the above example. The results are shown in Figure 13.

[0327] Example 14: Buffer capacity of Ab-hCD22 formulations - pH 4.0 to 6.0 Self-buffered formulations of Ab-hCD22 were prepared and titrated over the pH ranges 5.0-4.0 and 5.0-6.0 as described for Ab-hOPGL and Ab-hB7RP1 in the Examples above. Buffer capacity was calculated from the titration data as described above. Buffer capacity as a function of concentration is shown in FIG. 14 for both pH ranges. Panel A shows the buffer capacity of Ab-hCD22 formulations over the pH range 5.0-4.0. The buffer capacity is linearly dependent on concentration, with an approximately 21 mg / ml formulation of Ab-hCD22 having a buffer capacity equivalent to that of a similarly measured 10 mM sodium acetate buffer pH 5.0. Panel B shows the buffer capacity as a function of concentration over the pH range 5.0-6.0. Within this pH range, an approximately 30 mg / ml formulation of Ab-hCD22 has a buffer capacity equivalent to that of a similarly measured 10 mM sodium acetate buffer pH 5.0.

[0328] Example 15: Titration and buffer capacity of Ab-hIL4R formulations - pH 5.0 to 4.0 Self-buffering formulations of Ab-hIL4R were prepared at concentrations of 1, 10, 25, and 90 mg / ml as described for Ab-hOPGL in the above example. Titrations were performed with HCl as described above. Panel A of FIG. 15 shows the titration results. Panel B of FIG. 15 shows the dependence of buffering capacity on Ab-hIL4R concentration. The results clearly demonstrate the self-buffering capacity of Ab-hIL4R in this pH range. At approximately 75 mg / ml, the self-buffering formulation of Ab-hIL4R provides a buffering capacity in the titled pH range that is approximately the same as 10 mM sodium acetate pH 5.0, which was similarly measured.

[0329] Example 16: Titration and buffer capacity of Ab-hIL4R formulations - pH 5.0 to 6.0 Self-buffering formulations of Ab-hIL4R were prepared at concentrations of 1, 10, 25, and 90 mg / ml as described for Ab-hOPGL in the above example. Titrations were performed with NaOH as described above. Panel A of FIG. 16 shows the titration results. Panel B of FIG. 16 shows the dependence of buffering capacity on Ab-hIL4R concentration in this pH range. The results clearly demonstrate the self-buffering capacity of Ab-hIL4R in this pH range. At approximately 90 mg / ml, the self-buffering formulation of Ab-hIL4R provides a buffering capacity in the titled pH range that is approximately the same as that of 10 mM sodium acetate pH 5.0, which was similarly measured.

[0330] Example 17: Stability of Ab-hIL4R and pH in Acetate and Self-Buffered Ab-hIL4R Formulations at 37°C Self-buffered formulations of Ab-hIL4R at pH 5.0, 70 mg / ml and acetate buffer were prepared as described above. pH and Ab-hIL4R stability were monitored in the formulations for 4 weeks at 37° C. Ab-hIL4R stability was monitored by SE-HPLC as described above. The results are shown in FIG. 17. Panel A shows that Ab-hIL4R is at least as stable in the self-buffered formulation as Ab-hIL4R in the sodium acetate buffer formulation. Panel B shows that the pH in the self-buffered formulation is as stable as the pH in the sodium acetate buffer.

Claims

1. A composition comprising a pharmaceutical protein, wherein, at the pH of the composition, at 21°C, at one atmospheric pressure, and in equilibrium with the atmosphere, the protein has a buffer capacity per unit volume equal to or greater than the buffer capacity of about 4.0 mM sodium acetate buffer in pure water in the pH range of 5.0 to 4.0 or 5.0 to 5.5 under the same conditions, and further, the buffer capacity per unit volume of the composition under the same conditions excluding the buffer capacity of the protein is equal to or less than the buffer capacity per unit volume of 2.0 mM sodium acetate buffer in pure water in the pH range of 5.0 to 4.0 or 5.0 to 5.5 under the same conditions, and the composition has been approved for pharmaceutical use by an authority having legal authority to grant approval for pharmaceutical use.

2. A composition comprising a pharmaceutical protein, wherein at the pH of the composition, at 21°C, at atmospheric pressure, and in equilibrium with the atmosphere, the protein has a buffering capacity per unit volume of at least 1.50 mEq / liter-pH unit, and further, the buffering capacity per unit volume of the composition excluding said buffering capacity is less than 0.5 mEq / liter-pH unit, and the composition has been approved for pharmaceutical use by an authority with legal authority to grant approval for pharmaceutical use.

3. The composition of claim 1 , wherein the protein provides at least 80% of the buffering capacity of the composition.

4. The composition of claim 3, wherein the concentration of the protein is about 20 to 400 mg / ml.

5. The composition according to claim 4, wherein the pH maintained by the buffering action of the protein is about 3.5 to 8.

0.

6. The composition of claim 5, wherein the pH maintained by the buffering action of the protein is about 4 to 6.

7. 6. The composition of claim 5, further comprising one or more pharma- ceutically acceptable salts, wherein the total salt concentration is less than 150 mM.

8. 8. The composition of claim 7, further comprising one or more pharma- ceutically acceptable salts, wherein the total salt concentration is less than 100 mM.

9. The composition of claim 5 further comprising one or more pharma- ceutically acceptable polyols.

10. 10. The composition of claim 9, wherein the polyol is one or more of sorbitol, mannitol, sucrose, trehalose or glycerol.

11. The composition of claim 5 further comprising one or more pharma- ceutically acceptable surfactants.

12. 12. The composition of claim 11, wherein the surfactant is one or more of polysorbate 20, polysorbate 80, other fatty acid esters of sorbitan, polyethoxylates, and poloxamer 188.

13. 10. The composition of claim 9, further comprising one or more pharma- ceutically acceptable surfactants.

14. The composition of claim 1, further comprising one or more pharma- ceutically acceptable osmotic balancing agents; antioxidants; antibiotics; antifungals; bulking agents; lyoprotectants; antifoaming agents; chelating agents; preservatives; coloring agents; analgesics; or additional agents.

15. The composition of claim 5, further comprising one or more pharma- ceutically acceptable osmotic balancing agents; antioxidants; antibiotics; antifungals; bulking agents; lyoprotectants; antifoaming agents; chelating agents; preservatives; coloring agents; analgesics; or additional drugs.

16. The composition of claim 7, further comprising one or more pharma- ceutically acceptable: osmotic balancing agents; antioxidants; antibiotics; antifungals; bulking agents; lyoprotectants; antifoaming agents; chelating agents; preservatives; coloring agents; analgesics; or additional agents.

17. The protein is: an antibody, a Fab fragment, a Fab 2 Fragment, Fab 3 fragment, Fc fragment, scFv fragment, bis-scFv fragment, minibody, dimer, trimer, tetrabody, VhH domain, V-NAR domain, V H Domain, V L 15. The composition of any one of claims 1, 5, 7, 9, 11, 13 or 14, which is or comprises a domain, camelid Ig, Ig NAR, receptor, peptibody, or variant or derivative thereof or a protein related to these, or a variant thereof.

18. The composition of claim 17, wherein the protein comprises an Fc fragment or a portion thereof, or a variant or derivative of an Fc fragment or a portion thereof, or a protein related to an Fc fragment or a portion thereof, or a variant of any of these.

19. The composition of claim 18 , wherein the protein further comprises a first binding portion of a pair of cognate binding portions.

20. 15. The composition of any one of claims 1, 5, 7, 9, 11, 13 or 14, wherein the protein is selected from the group consisting of proteins that specifically bind to one or more of CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factors, fibroblast growth factors, transforming growth factors (TGFs), insulin-like growth factors, bone morphogenetic factors, insulin and insulin-related proteins, aggregation and aggregation-related proteins, colony stimulating factors (CSFs), other blood proteins and serum proteins blood group antigens; receptors, receptor-related proteins, growth hormone receptors, T cell receptors; neurotrophic factors, neurotrophins, relaxins, interferons, interleukins, viral antigens, lipoproteins, integrins, rheumatoid factors, immunotoxins, surface membrane proteins, transport proteins, homing receptors, addressins, regulatory proteins, and immunoadhesins.

21. 15. The composition of any one of claims 1, 5, 7, 9, 11, 13, or 14, wherein the protein is selected from the group consisting of an OPGL-specific binding protein, a myostatin-specific binding protein, an IL-4 receptor-specific binding protein, an IL1-R1-specific binding protein, an Ang2-specific binding protein, an NGF-specific binding protein, a CD22-specific binding protein, an IGF-1 receptor-specific binding protein, a B7RP-1-specific binding protein, an IFNγ-specific binding protein, a TALL-1-specific binding protein, a stem cell factor, an Flt-3 ligand, and an IL-17 receptor.

22. The proteins include CD3, CD4, CD8, CD19, CD20, CD34; HER2, HER3, HER4, EGF receptor; LFA-1, Mol, p150, 95, VLA-4, ICAM-1, VCAM, αv / β3 integrin; vascular endothelial growth factor ("VEGF"); growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, Mullerian inhibitory substance, human macrophage inflammatory protein (MIP-1-α), erythropoietin (EPO), NGF. -β, platelet-derived growth factor (PDGF), aFGF, bFGF, epidermal growth factor (EGF), TGF-α, TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, IGF-I, IGF-II, des(1-3)-IGF-I (brain IGF-I), insulin, insulin A chain, insulin B chain, proinsulin, insulin-like growth factor binding proteins; for example, in particular factor VIII, tissue factor, von Willebrand factor, protein C, α-1-antitrypsin, plasminogen activator, e.g. Urokinase and tissue plasminogen activator ("t-PA"), bombazine, thrombin, and thrombopoietin; M-CSF, GM-CSF, G-CSF, albumin, IgE, flk2 / flt3 receptor, obesity (OB) receptor, brain-derived neurotrophic factor (BDNF), NT-3, NT-4, NT-5, NT-6; relaxin A chain, relaxin B chain, prorelaxin; interferon-alpha, -beta, and -gamma; IL-1 through IL-10; AIDS enveloped viral antigen; calcitonin. The composition of any one of claims 1, 5, 7, 9, 11, 13 or 14, wherein the composition is selected from the group consisting of proteins that specifically bind to one or more of the following: tonin, glucagon, atrial natriuretic factor, pulmonary surfactant, tumor necrosis factor-alpha and -beta, enkephalinase, RANTES, mouse gonadotropin-related peptide, deoxyribonuclease (Dnase), inhibin, and activin; protein A or D, bone morphogenetic protein (BMP), superoxide dismutase, and decay accelerating factor (DAF).

23. The protein is Actimmune (interferon-gamma-1b), Activase (alteplase), Aldurazme (laronidase), Amevive (alefacept), Avonex (interferon beta-1a), BeneFIX (nonacog alfa), Beromun (tasonermin), Beatseron (interferon-beta-1b), BEXXAR (tositumomab), Tev-Tropin (somatropin), Bioclate or RECOMBINATE (recombinant), CEREZME (imiglucerase), ENBREL (etanercept), Eprex (epoetin alfa), EPOGEN / Procit (epoetin alfa), FABRAZYME (agalsidase beta), Fasturtec / Elitek ELITEK (rasburicase), FORTEO (teriparatide), GENOTROPIN (somatropin), GlucaGen (glucagon), Glucagon (glucagon, rDNA derived), GONAL-F (follitropin alfa), KOGENATE FS (ostocog alfa), HERCEPTIN (trastuzumab), HUMATROPE (somatropin), HUMIRA (adalimumab), insulin in solution, INFERGEN® (interferon alfacon-1), KINERET® (anakinra), Kogenate FS (antihemophilic factor), LEUKIN (sargramostim recombinant human granulocyte-macrophage colony-stimulating factor (rhuGM-CSF)), CAMPATH (alemtuzumab), RITUXAN® (rituximab), TNKase (tenecteplase), MYLOTARG (gemtuzumab) ozogamicin), NATRECOR (nesiritide), ARANESP (darbepoetin alfa), NEULASTA (pegfilgrastim), NEUMEGA (oprelvekin), NEUPOGEN (filgrastim), NORDITROPIN CARTRIDGES (somatropin), NOVOSEVEN (eptacog alfa), NUTROPIN AQ (somatropin), Oncaspar (pegaspargase), ONTAK (denileukin difitox), ORTHOCLONEOKT (muromonab-CD3), OVIDREL (choriogonadotropin alfa), PEGASYS (peginterferon alfa-2a), PROLEUKIN (aldesleukin), PULMOZYME (dornase alfa), Retavase (reteplase), REBETOL® (ribavirin) and REBETRON combination therapy including INTRON® A (interferon alfa-2b), REBIF (interferon beta-1a), REFACTO (antihemophilic factor), REFLUDAN (lepirudin), REMICADE (infliximab), REOPRO (abciximab) ROFERON®-A (interferon alfa-2 15. The composition of any one of claims 1, 5, 7, 9, 11, 13 or 14, wherein the medicament is selected from the group consisting of: a), SIMULECT (basiliximab), SOMAVERT (pegvisomant), SYNAGIS® (palivizumab), Stemben (ansestim, stem cell factor), THYROGEN, INTRON® A (interferon alpha-2b), PEG-INTRON® (peginterferon alpha-2b), XIGRIS® (activated drotrecogin alfa), XOLAIR® (omalizumab), ZENAPAX® (daclizumab), and ZEVALIN® (ibritumomab tiuxetan).

24. 15. The composition of any one of claims 1, 5, 7, 9, 11, 13 or 14, wherein the protein is Ab-hOPGL or a fragment thereof, or a mutant or derivative of Ab-hOPGL or a fragment thereof, or an Ab-hOPGL-related protein or a fragment thereof, or a variant of any of these.

25. The composition of any one of claims 1, 5, 7, 9, 11, 13 or 14, wherein the protein is Ab-hOPGL.

26. A composition according to any one of claims 1, 5, 7, 9, 11, 13 or 14, wherein the protein is Ab-hIL4R or a fragment thereof, or a mutant or derivative of Ab-hIL4R or a fragment thereof, or an Ab-hIL4R-related protein or a fragment thereof, or a variant of any of these.

27. The composition of any one of claims 1, 5, 7, 9, 11, 13 or 14, wherein the protein is Ab-hIL4R.

28. The composition of any one of claims 1, 5, 7, 9, 11, 13 or 14, wherein the protein is Ab-hB7RP1 or a fragment thereof, or a mutant or derivative of Ab-hB7RP1 or a fragment thereof, or an Ab-hB7RP1-related protein or a fragment thereof, or a variant of any of these.

29. The composition of any one of claims 1, 5, 7, 9, 11, 13 or 14, wherein the protein is Ab-hB7RP1.

30. A lyophilisate which upon reconstitution gives a composition according to any one of claims 1, 5, 7, 9, 11, 13 or 14.

31. 15. A kit comprising in one or more containers the composition of claim 1, 5, 7, 9, 11, 13 or 14 and instructions for its use.

32. 31. A kit comprising the lyophilisate of claim 30 and instructions for its use in one or more containers.

33. 17. A method of treating a subject comprising administering to the subject a composition according to any one of claims 1, 5, 7, 9, 11, 13 or 14 in a therapeutically effective amount and by a route.

34. 15. A method for preparing a composition according to any one of claims 1, 5, 7, 9, 11, 13 or 14, comprising removing residual buffer using a counterion.

35. 35. A method for preparing the composition of claim 34, comprising removing residual buffer using any one or more of size exclusion chromatography, dialysis, and / or cross-flow filtration in the presence of counterions.

36. 36. A method for preparing the composition of claim 35, comprising removing residual buffer using ion exchange chromatography.

37. 15. A method for preparing a composition according to any one of claims 1, 5, 7, 9, 11, 13 or 14, comprising removing residual buffer by diafiltration against an unbuffered solution having a pH below the desired pH.

38. 38. A method for producing a composition according to claim 37, wherein after diafiltration the pH is adjusted to the desired pH by addition of dilute acid and / or dilute base.

39. A lyophilisate which upon reconstitution provides the composition of claim 20.

40. 21. A kit comprising the composition of claim 20 and instructions for its use in one or more containers.

41. 41. A kit comprising the lyophilisate of claim 40 in one or more containers, and instructions for its use.

42. 21. A method of treating a subject comprising administering to the subject the composition of claim 20 in a therapeutically effective amount and by a route.

43. 21. A method for preparing the composition of claim 20 comprising removing residual buffer using a counterion.

44. 44. A method for preparing the composition of claim 43, comprising removing residual buffer using any one or more of size exclusion chromatography, dialysis, and / or cross-flow filtration in the presence of counterions.

45. 44. A method for preparing the composition of claim 43, comprising removing residual buffer using ion exchange chromatography.

46. 21. A method of making the composition of claim 20 comprising removing residual buffer by diafiltration against an unbuffered solution having a pH below the desired pH.

47. 47. A method for producing a composition according to claim 46, wherein after diafiltration the pH is adjusted to the desired pH by addition of dilute acid and / or dilute base.

48. A lyophilisate which upon reconstitution provides the composition of claim 21.

49. 22. A kit comprising the composition of claim 21 and instructions for its use in one or more containers.

50. 50. A kit comprising the lyophilisate of claim 49 and instructions for its use in one or more containers.

51. 22. A method of treating a subject comprising administering to the subject a composition of claim 21 in a therapeutically effective amount and by a route.

52. 22. A method for making the composition of claim 21 comprising removing residual buffer using a counterion.

53. 53. A method for preparing the composition of claim 52, comprising removing residual buffer using any one or more of size exclusion chromatography, dialysis, and / or cross-flow filtration in the presence of counterions.

54. 53. A method for preparing the composition of claim 52, comprising removing residual buffer using ion exchange chromatography.

55. 22. A method of making the composition of claim 21 comprising removing residual buffer by diafiltration against an unbuffered solution having a pH below the desired pH.

56. 56. A method for producing a composition according to claim 55, wherein after diafiltration the pH is adjusted to the desired pH by addition of dilute acid and / or dilute base.

57. A lyophilisate which upon reconstitution provides the composition of claim 23.

58. 24. A kit comprising the composition of claim 23 and instructions for its use in one or more containers.

59. 58. A kit comprising the lyophilisate of claim 57 in one or more containers, and instructions for its use.

60. 24. A method of treating a subject comprising administering to the subject the composition of claim 23 in a therapeutically effective amount and by a route.

61. 24. A method for preparing the composition of claim 23 comprising removing residual buffer using a counterion.

62. 62. A method for preparing the composition of claim 61, comprising removing residual buffer using any one or more of size exclusion chromatography, dialysis, and / or cross-flow filtration in the presence of counterions.

63. 62. A method for preparing the composition of claim 61, comprising removing residual buffer using ion exchange chromatography.

64. 62. A method of making the composition of claim 61 comprising removing residual buffer by diafiltration against an unbuffered solution having a pH below the desired pH.

65. 63. A method for producing a composition according to claim 62, wherein after diafiltration the pH is adjusted to the desired pH by addition of dilute acid and / or dilute base.

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