Self-buffering protein formulations   

This application is a continuation-in-part of and claims full priority benefit of U.S. Provisional Application Ser. No. 60/690,582 filed 14 Jun. 2005, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the formulation of proteins, especially pharmaceutical proteins. In particular, it relates to self-buffering biopharmaceutical protein compositions, and to methods for designing, making, and using the compositions. It further relates to pharmaceutical protein compositions for veterinary and/or for human medical use, and to methods relating thereto.

BACKGROUND OF THE INVENTION

Many aspects of pharmaceutical production and formulation processes are pH sensitive. Maintaining the correct pH of a finished pharmaceutical product is critical to its stability, effectiveness, and shelf life, and pH is an important consideration in designing formulations for administration that will be acceptable, as well as safe and effective.

To maintain pH, pharmaceutical processes and formulations use one or more buffering agents. A variety of buffering agents are available for pharmaceutical use. The buffer or buffers for a given application must be effective at the desired pH. They must also provide sufficient buffer capacity to maintain the desired pH for as long as necessary. A good buffer for a pharmaceutical composition must satisfy numerous other requirements as well. It must be appropriately soluble. It must not form deleterious complexes with metal ions, be toxic, or unduly penetrate, solubilize, or absorb on membranes or other surfaces. It should not interact with other components of the composition in any manner which decreases their availability or effectiveness. It must be stable and effective at maintaining pH over the range of conditions to which it will be exposed during formulation and during storage of the product. It must not be deleteriously affected by oxidation or other reactions occurring in its environment, such as those that occur in the processing of the composition in which it is providing the buffering action. If carried over or incorporated into a final product, a buffering agent must be safe for administration, compatible with other components of the composition over the shelf-life of the product, and acceptable for administration to the end user.

Although there are many buffers in general use, only a limited number are suitable for biological applications and, of these, fewer still are acceptable for pharmaceutical processes and formulations. As a result, it often is challenging to find a buffer that not only will be effective at maintaining pH but also will meet all the other requirements for a given pharmaceutical process, formulation, or product.

The challenge of finding a suitable buffer for pharmaceutical use can be especially acute for pharmaceutical proteins. The conformation and activity of proteins are critically dependent upon pH. Proteins are susceptible to a variety of pH sensitive reactions that are deleterious to their efficacy, typically many more than affect small molecule drugs. For instance, to mention just a few salient examples, the side chain amides of asparagine and glutamine are deamidated at low pH (less than 4.0) and also at neutral or high pH (greater than 6.0). Aspartic acid residues promote the hydrolysis of adjacent peptide bonds at low pH. The stability and disposition of disulfide bonds is highly dependent on pH, particularly in the presence of thiols. Solubility, flocculation, aggregation, precipitation, and fibrillation of proteins are critically dependent on pH. The crystal habit important to some pharmaceutical formulations also is critically dependent on pH. And pH is also an important factor in surface adsorption of many pharmaceutical peptides and proteins.

Buffering agents that catalyze reactions that inactivate and/or degrade one or more other ingredients, moreover, cannot be used in pharmaceutical formulations. Buffers for pharmaceutical use must have not only the buffer capacity required to maintain correct pH, but also they must not buffer so strongly that their administration deleteriously perturbs a subject\'s physiological pH. Buffers for pharmaceutical formulations also must be compatible with typically complex formulation processes. For instance, buffers that sublime or evaporate, such as acetate and imidazole, generally cannot be relied upon to maintain pH during lyophilization and in the reconstituted lyophilization product. Other buffers that crystallize out of the protein amorphous phase, such as sodium phosphate, cannot be relied upon to maintain pH in processes that require freezing.

Buffers used to maintain pH in pharmaceutical end-products also must be not only effective at maintaining pH but also safe and acceptable for administration to the subject. For instance, several otherwise useful buffers, such as citrate at low or high concentration and acetate at high concentration, are undesirably painful when administered parenterally.

Some buffers have been found to be useful in the formulation of pharmaceutical proteins, such as acetate, succinate, citrate, histidine (imidazole), phosphate, and Tris. They all have undesirable limitations and disadvantages. And they all have the inherent disadvantage of being an additional ingredient in the formulation, which complicates the formulation process, poses a risk of deleteriously affecting other ingredients, stability, shelf-life, and acceptability to the end user.

There is a need, therefore, for additional and improved methods of maintaining pH in the production and formulation of pharmaceuticals and in pharmaceutical compositions, particularly in the production and formulation of biopharmaceutical proteins and in biopharmaceutical protein compositions.

SUMMARY

Therefore, it is among the various objects and aspects of the invention to provide, in certain of the preferred embodiments, protein formulations comprising a protein, particularly pharmaceutically acceptable formulations comprising a pharmaceutical protein, that are buffered by the protein itself, that do not require additional buffering agents to maintain a desired pH, and in which the protein is substantially the only buffering agent (i.e., other ingredients, if any, do not act substantially as buffering agents in the formulation).

In this regard and others, it is among the various objects and aspects of the invention to provide, in certain preferred embodiments, self-buffering formulations of a protein, particularly of a pharmaceutical protein, characterized in that the concentration of the formulated protein provides a desired buffer capacity.

It is further among the various objects and aspects of the invention to provide, in certain of the particularly preferred embodiments, self-buffering protein formulations, particularly pharmaceutical protein formulations, in which the total salt concentration is less than 150 mM.

It is further among the various objects and aspects of the invention to provide, in certain of the particularly preferred embodiments, self-buffering protein formulations, particularly pharmaceutical protein formulations, that further comprise one or more polyols and/or one or more surfactants.

It is also further among the various objects and aspects of the invention to provide, in certain of the particularly preferred embodiments, self-buffering formulations comprising a protein, particularly a pharmaceutical protein, in which the total salt concentration is less than 150 mM, that further comprise one or more excipients, including but not limited to, pharmaceutically acceptable salts; osmotic balancing agents (tonicity agents); surfactants, polyols, anti-oxidants; antibiotics; antimycotics; bulking agents; lyoprotectants; anti-foaming agents; chelating agents; preservatives; colorants; and analgesics.

It is additionally among the various objects and aspects of the invention to provide, in certain preferred embodiments, self-buffering protein formulations, particularly pharmaceutical protein formulations, that comprise, in addition to the protein, one or more other pharmaceutically active agents.

Various additional aspects and embodiments of the invention are illustratively described in the following numbered paragraphs. The invention is described by way of reference to each of the items set forth in the paragraphs, individually and/or taken together in any combination. Applicant specifically reserves the right to assert claims based on any such combination.

1. A composition according to any of the following, wherein the composition has been approved for pharmaceutical use by a national or international authority empowered by law to grant such approval preferably the European Agency for the Evaluation of Medical Products, Japan\'s Ministry of Health, Labor and Welfare, China\'s State Drug Administration, United States Food and Drug Administration, or their successor(s) in this authority, particularly preferably the United States Food and Drug Administration or its successor(s) in this authority.

2. A composition according to any of the foregoing or the following, wherein the composition is produced in accordance with good manufacturing practices applicable to the production of pharmaceuticals for use in humans.

3. A composition according to any of the foregoing or the following, comprising a protein, the protein having a buffer capacity per unit volume per pH unit of at least that of approximately: 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 sodium acetate buffer in pure water over the range of pH 5.0 to 4.0 or pH 5.0 to 5.5, preferably as determined in accordance with the methods described in Example 1 and 2, particularly preferably at least 2.0 mM, especially particularly preferably at least 3.0 mM, very especially particularly preferably at least 4.0 mM or at least 5.0 mM, especially particularly preferably at least 7.5 mM, particularly preferably at least 10 mM, preferably at least 20 mM.

4. A composition according to any of the foregoing or the following wherein, exclusive of the buffer capacity of the protein, the buffer capacity per unit volume per pH unit of the composition is equal to or less than that 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 range of pH 4.0 to 5.0 or pH 5.0 to 5.5, preferably as determined in accordance with the methods described in Example 1 and 2, particularly preferably less than that of 1.0 mM, very especially particularly preferably less than that of 2.0 mM, especially particularly preferably less than that of 2.5 mM, particularly preferably less than that of 3.0 mM, preferably less than that of 5.0 mM.

5. A composition according to any of the foregoing or the following comprising a protein wherein over the range of plus or minus 1 pH unit from the pH of the composition, the buffer capacity of the protein is at least approximately: 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 per liter per pH unit, preferably at least approximately 1.00, particularly preferably 1.50, especially particularly preferably 1.63, very especially particularly preferably 2.00, very highly especially particularly preferably 3.00, very especially particularly preferably 5.0, especially particularly preferably 10.0, particularly preferably 20.0.

6. A composition according to any of the foregoing or the following comprising a protein wherein over the range of plus or minus 1 pH unit from the pH of the composition, exclusive of the protein, the buffer capacity per unit volume per pH unit of the composition is equal to or less than that 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 range pH 5.0 to 4.0 or pH 5.0 to 5.5, particularly preferably determined in accordance with Example 1 and/or Example 2.

7. A composition according to any of the foregoing or the following, wherein over a range of plus or minus 1 pH unit from a desired pH, the protein provides at least approximately 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of the buffer capacity of the composition, preferably at least approximately 75%, particularly preferably at least approximately 85%, especially particularly preferably at least approximately 90%, very especially particularly preferably at least approximately 95%, very highly especially particularly preferably at least approximately 99% of the buffer capacity of the composition.

8. A composition according to any of the foregoing or the following, wherein the concentration of the protein is between approximately: 20 and 400, or 20 and 300, or 20 and 250, or 20 and 200, or 20 and 150 mg/ml, preferably between approximately 20 and 400 mg/ml, particularly preferably between approximately 20 and 250, especially particularly between approximately 20 and 150 mg/ml.

9. A composition according to any of the foregoing or the following, wherein the pH maintained by the buffering action of the protein is between approximately: 3.5 and 8.0, or 4.0 and 6.0, or 4.0 and 5.5, or 4.0 and 5.0, preferably between approximately 3.5 and 8.0, especially particularly preferably approximately 4.0 and 5.5.

10. A composition according to any of the foregoing or the following, 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, especially preferably 100 mM, very particularly preferably 75 mM, particularly preferably 50 mM, preferably 25 mM.

11. A composition according to any of the foregoing or the following, further comprising one or more pharmaceutically acceptable salts; polyols; surfactants; osmotic balancing agents; tonicity agents; anti-oxidants; antibiotics; antimycotics; bulking agents; lyoprotectants; anti-foaming agents; chelating agents; preservatives; colorants; analgesics; or additional pharmaceutical agents.

12. A composition according to any of the foregoing or the following, comprising one or more pharmaceutically acceptable polyols in an amount that is hypotonic, isotonic, or hypertonic, preferably approximately isotonic, particularly preferably isotonic, especially preferably any one or more of sorbitol, mannitol, sucrose, trehalose, or glycerol, particularly especially preferably approximately 5% sorbitol, 5% mannitol, 9% sucrose, 9% trehalose, or 2.5% glycerol, very especially in this regard 5% sorbitol, 5% mannitol, 9% sucrose, 9% trehalose, or 2.5% glycerol.

13. A composition according to any of the foregoing or the following, 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 approximately 0.001 to 0.1% polysorbate 20 or polysorbate 80, very preferably approximately 0.002 to 0.02% polysorbate 20 or polysorbate 80, especially 0.002 to 0.02% polysorbate 20 or polysorbate 80.

14. A composition according to any of the foregoing or the following, wherein the protein is a pharmaceutical agent and the composition is a sterile formulation thereof suitable for treatment of a non-human or a human subject.

15. A composition according to any of the foregoing or the following, wherein the protein is a pharmaceutical agent effective to treat a disease and the composition is a sterile formulation thereof suitable for administration to a subject for treatment thereof.

16. A composition according to any of the foregoing or the following, wherein the protein does not induce a significantly deleterious antigenic response following administration to a subject.

17. A composition according to any of the foregoing or the following, wherein the protein does not induce a significantly deleterious immune response following administration to a subject.

18. A composition according to any of the foregoing or the following, wherein the protein is a human protein.

19. A composition according to any of the foregoing or the following, wherein the protein is a humanized protein.

20. A method according to any of the foregoing or the following, 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, especially an IgG2 antibody.

21. A composition according to any of the foregoing or the following, wherein the protein comprises a: Fab fragment, Fab2 fragment, Fab3 fragment, Fc fragment, scFv fragment, bis-scFv(s) fragment, minibody, diabody, triabody, tetrabody, VhH domain, V-NAR domain, VH domain, VL domain, camel Ig, Ig NAR, or peptibody, or a variant, derivative, or modification of any of the foregoing.

22. A composition according to any of the foregoing or the following, wherein the protein comprises an Fc fragment or a part thereof or a derivative or variant of an Fc fragment or part thereof.

23. A composition according to any of the foregoing or the following, wherein the protein comprises a first binding moiety of a pair of cognate binding moieties, wherein the first moiety binds the second moiety specifically.

24. A composition according to any of the foregoing or the following, wherein the protein comprises (a) an Fc fragment or a part thereof or a derivative or variant of an Fc fragment or part thereof, and (b) a first binding moiety of a pair of cognate binding moieties.

25. A composition according to any of claim 1, 5, 7, 9, 11, 13, or 14, wherein the protein is selected from the group consisting of proteins that bind specifically to one or more CD proteins, HER receptor family proteins, cell adhesion molecules, growth factors, nerve growth factors, fibroblast growth factors, transforming growth factors (TGF), insulin-like growth factors, osteoinductive factors, insulins and insulin-related proteins, coagulation and coagulation-related proteins, colony stimulating factors (CSFs), other blood and serum proteins blood group antigens; receptors, receptor-associated 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,

26. A composition according to any of the foregoing or the following, wherein the protein is selected from the group consisting of OPGL specific binding proteins, myostatin specific binding proteins, IL-4 receptor specific binding proteins, IL1-R1 specific binding proteins, Ang2 specific binding proteins, NGF-specific binding proteins, CD22 specific binding proteins, IGF-1 receptor specific binding proteins, B7RP-1 specific binding proteins, IFN gamma specific binding proteins, TALL-1 specific binding proteins, stem cell factors, Flt-3 ligands, and IL-17 receptors.

27. A composition according to any of the foregoing or the following, wherein the protein is selected from the group consisting of proteins that bind specifically to one ormore of: CD3, CD4, CD8, CD19, CD20, CD34; HER2, HER3, HER4, the EGF receptor; LFA-1, Mol, p150,95, VLA-4, ICAM-1, VCAM, alpha v/beta 3 integrin; vascular endothelial growth factor (“VEGF”); growth hormone, thyroid stimulating hormone, follicle stimulating hormone, luteinizing hormone, growth hormone releasing factor, parathyroid hormone, mullerian-inhibiting substance, human macrophage inflammatory protein (MIP-1-alpha), erythropoietin (EPO), NGF-beta, platelet-derived growth factor (PDGF), aFGF, bFGF, epidermal growth factor (EGF), TGF-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, 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; such as, among others, factor VIII, tissue factor, von Willebrands factor, protein C, alpha-1-antitrypsin, plasminogen activators, such as 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, bone-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 to IL-10; AIDS envelope viral antigen; calcitonin, glucagon, atrial natriuretic factor, lung surfactant, tumor necrosis factor-alpha and -beta, enkephalinase, RANTES, mouse gonadotropin-associated peptide, Dnase, inhibin, and activin; protein A or D, bone morphogenetic protein (BMP), superoxide dismutase, decay accelerating factor (DAF).

28. A composition according to any of the foregoing or the following, wherein the protein is selected from the group consisting of: 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 alpha), EPOGEN/Procit (Epoetin alfa), FABRAZYME (Agalsidase beta), Fasturtec/Elitek ELITEK (Rasburicase), FORTEO (Teriparatide), GENOTROPIN (Somatropin), GlucaGen (Glucagon), Glucagon (Glucagon, rDNA origin), GONAL-F (follitropin alfa), KOGENATE FS (Octocog 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 diftitox), ORTHOCLONE OKT (muromonab-CD3), OVIDREL (choriogonadotropin alfa), PEGASYS (peginterferon alfa-2a), PROLEUKIN (Aldesleukin), PULMOZYME (dornase alfa), Retavase (Reteplase), REBETRON Combination Therapy containing REBETOL® (Ribavirin) and INTRONS A (Interferon alfa-2b), REBIF (interferon beta-1a), REFACTO (Antihemophilic Factor), REFLUDAN (lepirudin), REMICADE (infliximab), REOPRO (abciximab)ROFERON®-A (Interferon alfa-2a), SIMULECT (baasiliximab), SOMAVERT (Pegivisomant), SYNAGIS® (palivizumab), Stemben (Ancestim, Stem cell factor), THYROGEN, INTRON® A (Interferon alfa-2b), PEG-INTRON® (Peginterferon alfa-2b), XIGRIS® (Drotrecogin alfa activated), XOLAIR® (Omalizumab), ZENAPAX® (daclizumab), and ZEVALIN® (Ibritumomab Tiuxetan).

29. A composition according to any of the foregoing or the following, wherein the protein is Ab-hCD22 or a fragment thereof, or a variant, derivative, or modification of Ab-hCD22 or of a fragment thereof; Ab-hIL4R or a fragment thereof, or a variant, derivative, or modification of Ab-hIL4R or of a fragment thereof; Ab-hOPGL or a fragment thereof, or a variant, derivative, or modification of Ab-hOPGL or of a fragment thereof, or Ab-hB7RP1 or a fragment thereof, or a variant, derivative, or modification of Ab-hB7RP1 or of a fragment thereof.

30. A composition according to any of the foregoing or the following, wherein the protein is: Ab-hCD22 or Ab-hIL4R or Ab-hOPGL or Ab-hB7RP1.

31. A composition according to any of the foregoing or the following comprising a protein and a solvent, the protein having a buffer capacity per unit volume per pH unit of at least that of 4.0 mM sodium acetate in water over the range of pH 4.0 to 5.0 or pH 5.0 to 5.5, particularly as determined by the methods described in Examples 1 and 2, wherein the buffer capacity per unit volume of the composition exclusive of the protein is equal to or less than that of 2.0 mM sodium acetate in water over the same ranges preferably determined in the same way.

32. A composition according to any of the foregoing or the following comprising a protein and a solvent, wherein at the pH of the composition the buffer capacity of the protein is at least 1.63 mEq per liter for a pH change of the composition of plus or minus 1 pH unit wherein the buffer capacity of the composition exclusive of the protein is equal to or less than 0.81 mEq per liter at the pH of the composition for a pH change of plus or minus 1 pH unit.

33. A lyophilate which upon reconstitution provides a composition in accordance with any of the foregoing or the following.

34. A kit comprising in one or more containers a composition or a lyophilate in accordance with any of the foregoing or the following, and instructions regarding use thereof.

35. A process for preparing a composition or a lyophilate according to any of the foregoing or the following, comprising removing residual buffer using a counter ion.

36. A process for preparing a composition or a lyophilate according to any of the foregoing or the following, comprising removing residual buffer using any one or more of the following in the presence of a counter ion: chromatography, dialysis, and/or tangential flow filtration.

37. A process for preparing a composition or a lyophilate according to any of the foregoing or the following, comprising removing residual buffer using tangential flow filtration.

38. A process for preparing a composition or a lyophilate according to any of the foregoing or the following comprising a step of dialysis against a solution at a pH below that of the preparation, and, if necessary, adjusting the pH thereafter by addition of dilute acid or dilute base.

39. A method for treating a subject comprising administering to a subject in an amount and by a route effective for treatment a composition according to any of the foregoing or the following, including a reconstituted lyophilate.

FIG. 1 depicts titration data and buffer capacity as a function of concentration for sodium acetate standard buffers over the range from pH 5.0 to 4.0. Panel A is a graph that depicts the pH change upon acid titration of several different concentrations of a standard sodium acetate buffer, as described in Example 1. pH is indicated on the vertical axis. The amount of acid added to each solution is indicated on the horizontal axis in microequivalents of HCl added per ml of solution (μEq/ml). The linear least squares trend lines are depicted for each dataset. Acetate concentrations are indicated in the inset. Panel B is a graph that depicts the buffer capacity of the acetate buffers over the acidic pH range as determined from the titration data depicted in Panel A, as described in Example 1. Buffer capacity is indicated on the vertical axis as microequivlents of acid per ml of buffer solution per unit change in pH (μEq/ml-pH). Acetate concentration is indicated on the horizontal axis in mM.

FIG. 2 depicts titration data and buffer capacity as a function of concentrations for sodium acetate standard buffers over the range from pH 5.0 to 5.5. Panel A is a graph that depicts the pH change upon base titration of several different concentration of a standard sodium acetate buffer, as described in Example 2. pH is indicated on the vertical axis. The amount of base added to each solution is indicated on the horizontal axis in microequivalents of NaOH added per ml of solution (μEq/ml). The linear least squares trend lines are depicted for each dataset. Acetate concentrations are indicated in the inset. Panel B is a graph that depicts the buffer capacity of the acetate buffers over the basic pH range as determined from the titration data depicted in Panel A and described in Example 2. Buffer capacity is indicated on the vertical axis as microequivlents of base per ml of buffer solution per unit change in pH (μEq/ml-pH). Acetate concentration is indicated on the horizontal axis in mM.

FIG. 3 depicts the determination of acetate concentration in acetate buffer standards, as described in Example 3. The graph shows a standard curve for the determinations, with peak area indicated on the vertical axis and the acetate concentration indicated on the horizontal axis. The nominal and the measured amounts of acetate in the solutions used for the empirical determination of buffer capacity are tabulated below the graph.

FIG. 4 is a graph that depicts the pH change upon acid titration of several different concentrations of Ab-hOPGL over the range of pH 5.0 to 4.0, as described in Example 4. pH is indicated on the vertical axis. The amount of acid added to the solutions is indicated on the horizontal axis in microequivalents of HCl added per ml of buffer solution (μEq/ml). The linear least squares trend lines are depicted for each dataset. Ab-hOPGL concentrations are indicated in the inset.

FIG. 5 is a graph that depicts the pH change upon base titration of several different concentrations of Ab-hOPGL over the range 5.0 to 6.0, as described in Example 5. pH is indicated on the vertical axis. The amount of base added to the solutions is indicated on the horizontal axis in microequivalents of NaOH added per ml of buffer solution (μEq/ml). The linear least squares trend lines are depicted for each dataset. Ab-hOPGL concentrations are indicated in the inset.

FIG. 6 shows the residual acetate levels in Ab-hOPGL solutions used for determining buffer capacity. The graph shows the standard curve used for the acetate determinations as described in Example 6. The nominal and the experimentally measured acetate concentrations in the solutions are tabulated below the graph.

FIG. 7 is a graph depicting the buffer capacity of Ab-hOPGL plus or minus residual acetate in the pH range 5.0 to 4.0. The data were obtained as described in Example 7. The upper line shows Ab-hOPGL buffer capacity with residual acetate. The lower line shows Ab-hOPGL buffer capacity adjusted for residual acetate. The vertical axis indicates buffer capacity in microequivalents of acid per ml of Ab-hOPGL solution per unit of pH (μEq/ml-pH). The horizontal axis indicates the concentration of Ab-hOPGL in mg/ml. The buffer capacities of different concentrations of standard acetate buffers as described in Example 1 are shown as horizontal lines. The concentrations of the buffers are indicated above the lines.

FIG. 8 is a graph depicting the buffer capacity of Ab-hOPGL plus or minus residual acetate in the basic pH range pH 5.0 to 6.0. The data were obtained as described in Example 8. The upper line depicts Ab-hOPGL buffer capacity with residual acetate. The lower line depicts Ab-hOPGL buffer capacity adjusted for residual acetate. The vertical axis indicates buffer capacity in microequivalents of base added per ml of buffer solution per unit of pH (μEq/ml-pH). The horizontal axis indicates the concentration of Ab-hOPGL in mg/ml. The buffer capacities of several concentrations of standard sodium acetate buffers as described in Example 2 are indicated by horizontal lines, The acetate concentrations are indicated above each line.

FIG. 9 depicts, in a pair of charts, pH and Ab-hOPGL stability in self-buffering and conventionally buffered formulations. Panel A depicts the stability of self-buffered Ab-hOPGL, Ab-hOPGL formulated in acetate buffer, and Ab-hOPGL formulated in glutamate as a function of storage time at 4° C. over a period of six months. The vertical axis indicates Ab-hOPGL stability in percent Ab-hOPGL monomer determined by SE-HPLC. Storage time is indicated on the horizontal axis. Panel B depicts the pH of the same three formulations measured over the same period of time. The determinations of protein stability and the measurements of pH are described in Example 9.

FIG. 10 depicts titration curves and buffer capacities for several concentrations of self-buffering Ab-hB7RP1 formulations over the range of pH 5.0 to 4.0. Panel A shows the titration data. pH is indicated on the vertical axis. The amount of acid added to the solutions is indicated on the horizontal axis in microequivalents of HCl added per ml of buffer solution (μEq/ml). The linear least squares trend lines are depicted for each dataset. The Ab-hB7RP1 concentrations are indicated in the inset. Panel B depicts the buffer capacities of Ab-hB7RP1 formulations. The upper line shows the buffer capacities for the formulations including the contribution of residual acetate. The lower line shows the buffer capacities for formulations after subtracting the contribution of residual acetate based on SE-HPLC determinations as described in Example 3. Linear least squares trend lines are shown for the two data sets. The vertical axis indicates buffer capacity in microequivalents of acid per ml of buffer solution per unit of pH (μEq/ml-pH). The concentration of Ab-hB7RP1 is indicated on the horizontal axis in mg/ml. The buffer capacities of several concentrations of standard sodium acetate buffers as described in Example 1 are shown by dashed horizontal lines. The acetate buffer concentration are shown below each line. The results were obtained as described in Example 10.

FIG. 11 depicts titration curves and buffer capacities for several concentrations of self-buffering Ab-hB7RP1 formulations over the range of pH 5.0 to 6.0. Panel A shows the titration data. pH is indicated on the vertical axis. The amount of base added to the solutions is indicated on the horizontal axis in microequivalents of NaOH added per ml of buffer solution (μEq/ml). The linear least squares trend lines are depicted for each dataset. The Ab-hB7RP1 concentrations are indicated in the inset. Panel B depicts the buffer capacities of Ab-hB7RP1 formulations. The upper line shows the buffer capacities for the formulations containing residual acetate. The lower line shows the buffer capacities for formulations adjusted to remove the contribution of residual acetate. Linear least squares trend lines are shown for the two data sets. The vertical axis indicates buffer capacity in microequivalents of base per ml of buffer solution per unit of pH (μEq/ml-pH). The concentration of Ab-hB7RP1 is indicated on the horizontal axis in mg/ml. The buffer capacities of several concentrations of standard sodium acetate buffers as described in Example 2 are shown by dashed horizontal lines. The acetate buffer concentrations are shown above each line. The results were obtained as described in Example 11.

FIG. 12 depicts Ab-hB7RP1 stability in self-buffering 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 at 4° C. over a period of six months. The vertical axis depicts Ab-hB7RP1 monomer in the samples determined by SE-HPLC. Time is indicated on the horizontal axis. Panel B depicts the stability of the same three formulations as a function of storage at 29° C. over the same period of time. Axes in Panel B are the same as in Panel A. The determinations of protein stability by HPLC-SE are described in Example 12.

FIG. 13 depicts pH stability in self buffer formulations of Ab-hB7RP1 at 4° C. and 29° C. The vertical axis indicates pH. Time, in weeks, is indicated on the horizontal axis. Temperatures of the datasets are indicated in the inset. The data were obtained as described in Example 13.

FIG. 14 depicts the buffer capacity of self-buffering formulations of Ab-hCD22 as a function of Ab-hCD22 concentration over the range of pH 4.0 to 6.0. Panel A depicts the buffer capacities of self-buffering Ab-hCD22 formulations as a function of Ab-hCD22 concentration over the range of pH 4.0 to 5.0. Panel B depicts the buffer capacities of self-buffering Ab-hCD22 formulations as a function of concentration over the range of pH 5.0 to 6.0. In both panels the vertical axis indicates buffer capacity in microequivalents of base per ml of buffer solution per unit of pH (μEq/ml-pH), and the horizontal axis indicates Ab-hCD22 concentrations in mg/ml. For reference, the buffer capacity of 10 mM sodium acetate as described in Example 1 is shown in both panels by a dashed horizontal line. The results shown in the Figure were obtained as described in Example 14.

FIG. 15 depicts titration curves and buffer capacities for several concentrations of self-buffering Ab-hIL4R formulations over the range of pH 5.0 to 4.0. Panel A shows the titration data. pH is indicated on the vertical axis. The amount of acid added to the solutions is indicated on the horizontal axis in microequivalents of HCl added per ml of buffer solution (μEq/ml). The linear least squares trend lines are depicted for each dataset. The Ab-hIL4R concentrations are indicated in the inset. Panel B depicts the buffer capacities of Ab-hIL4R as a function of concentration. The linear least squares trend line is shown for the dataset. The vertical axis indicates buffer capacity in microequivalents of base per ml of buffer solution per unit of pH (μEq/ml-pH). The concentration of Ab-hIL4R is indicated on the horizontal axis in mg/ml. The buffer capacities of several concentrations of standard sodium acetate buffers as described in Example 1 are shown by dashed horizontal lines. The acetate buffer concentrations are shown above each line. The results were obtained as described in Example 15.

FIG. 16 depicts titration curves and buffer capacities for several concentrations of self-buffering Ab-hIL4R formulations over the range of pH 5.0 to 6.0. Panel A shows the titration data. pH is indicated on the vertical axis. The amount of base added to the solutions is indicated on the horizontal axis in microequivalents of NaOH added per ml of buffer solution (μEq/ml). The linear least squares trend lines are depicted for each dataset. The Ab-hIL4R concentrations are indicated in the inset. Panel B depicts the buffer capacities of Ab-hIL4R as a function of concentration. The linear least squares trend line is shown for the dataset. The vertical axis indicates buffer capacity in microequivalents of base per ml of buffer solution per unit of pH (μEq/ml-pH). The concentration of Ab-hIL4R is indicated on the horizontal axis in mg/ml. The buffer capacities of several concentrations of standard sodium acetate buffers as described in Example 2 are shown by dashed horizontal lines. The acetate buffer concentrations are shown above each line. The results were obtained as described in Example 16.

FIG. 17 depicts Ab-hIL4R and pH stability in acetate buffered and self-buffered formulations of Ab-hIL4R at 37° C. as a function of time. Panel A is a bar graph showing Ab-hIL4R stability over four weeks at 37° C. The vertical axis indicates stability in percent monomeric Ab-hIL4R as determined by SE-HPLC. The horizontal axis indicates storage time in weeks. The insert identifies the data for the acetate and for the self-buffered formulations. Panel B shows the pH stability of the same formulations for the same conditions and time periods. The pH is indicated on the vertical axis. Storage time in weeks is indicated on the horizontal axis. Data for the acetate and self-buffered formulations are indicated in the inset. The data were obtained as described in Example 17.

The meanings ascribed to various terms and phrases as used herein are illustratively explained below.

“A” or “an” herein means “at least one;” “one or more than one.”

“About,” unless otherwise stated explicitly herein, means V 20%. For instance about 100 herein 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%).

“Agonist(s)” means herein a molecular entity that is different from a corresponding stimulatory ligand but has the same stimulatory effect. For instance (although agonists work through other mechanisms), for a hormone that stimulates an activity by binding to a corresponding hormone receptor, an agonist is a chemically different entity that binds the hormone receptor and stimulates its activity.

“Antagonist(s)” means herein a molecular entity that is different from a corresponding ligand and has an opposite effect. For instance (although antagonists work through other mechanisms), one type of antagonist of a hormone that stimulates an activity by binding to a corresponding hormone receptor is a chemical entity that is different from the hormone and binds the hormone receptor but does not stimulate the activity engendered by hormone binding, and by this action inhibits the effector activity of the hormone.

“Antibody(s)” is used herein in accordance with its ordinary meaning in the biochemical and biotechnological arts.

Among antibodies within the meaning of the term as it is used herein, are those isolated from biological sources, including monoclonal and polyclonal antibodies, antibodies made by recombinant DNA techniques (also referred to at times herein as recombinant antibodies), including those made by processes that involve activating an endogenous gene and those that involve expression of an exogenous expression construct, including antibodies made in cell culture and those made in transgenic plants and animals, and antibodies made by methods involving chemical synthesis, including peptide synthesis and semi-synthesis. Also within the scope of the term as it is used herein, except as otherwise explicitly set forth, are chimeric antibodies and hybrid antibodies, among others.

The prototypical antibody is a tetrameric glycoprotein comprised of two identical light chain-heavy chain dimers joined together by disulfide bonds. There are two types of vertebrate light chains, kappa and lambda. Each light chain is comprised of a constant region and a variable region. The two light chains are distinguished by constant region sequences. There are five types of vertebrate heavy chains: alpha, delta, epsilon, gamma, and mu. Each heavy chain is comprised of a variable region and three constant regions. The five heavy chain types define five classes of vertebrate antibodies (isotypes): IgA, IgD, IgE, IgG, and IgM. Each isotype is made up of, respectively, (a) two alpha, delta, epsilon, gamma, or mu heavy chains, and (b) two kappa or two lambda light chains. The heavy chains in each class associate with both types of light chains; but, the two light chains in a given molecule are both kappa or both lambda. IgD, IgE, and IgG generally occur as “free” heterotetrameric glycoproteins. IgA and IgM generally occur in complexes comprising several IgA or several IgM heterotetramers associated with a “J” chain polypeptide. Some vertebrate isotypes are classified into subclasses, distinguished from one another by differences in constant region sequences. There are four human IgG subclasses, IgG1, IgG2, IgG3, and IgG4, and two IgA subclasses, IgA1 and IgA2, for example. All of these and others not specifically described above are included in the meaning of the term “antibody(s)” as used herein.

The term “antibody(s)” further includes amino acid sequence variants of any of the foregoing as described further elsewhere herein.

“Antibody-derived” as used herein means any protein produced from an antibody, and any protein of a design based on an antibody. The term includes in its meaning proteins produced using all or part of an antibody, those comprising all or part of an antibody, and those designed in whole or in part on the basis of all or part of an antibody. “Antibody-derived” proteins include, but are not limited to, Fc, Fab, and Fab2 fragments and proteins comprising the same, VH domain and VL domain fragments and proteins comprising the same, other proteins that comprise a variable and/or a constant region of an antibody, in whole or in part, scFv(s) intrabodies, maxibodies, minibodies, diabodies, amino acid sequence variants of the foregoing, and a variety of other such molecules, including but not limited to others described elsewhere herein.

“Antibody-related” as used herein means any protein or mimetic resembling in its structure, function, or design an antibody or any part of an antibody. Among “antibody-related” proteins as the term is used herein are “antibody-derived” proteins as described above. It is to be noted that the terms “antibody-derived” and “antibody-related” substantially overlap; both terms apply to many such proteins. Examples of “antibody-related” proteins, without implying limitation in this respect, are peptibodies and receptibodies. Other examples of “antibody-related” proteins are described elsewhere herein.

“Antibody polypeptide(s)” as used herein, except as otherwise noted, means a polypeptide that is part of an antibody, such as a light chain polypeptide, a heavy chain polypeptide and a J chain polypeptide, to mention a few examples, including among others fragments, derivatives, and variants\' thereof, and related polypeptides.

“Approximately” unless otherwise noted means the same as about.

“Binding moiety(s)” means a part of a molecule or a complex of molecules that binds specifically to part of another molecule or complex of molecules. The binding moiety may be the same or different from the part of the molecule or complex of molecules to which it binds. The binding moiety may be all of a molecule or complex of molecules as well.

“Binds specifically” is used herein in accordance with its ordinary meaning in the art and means, except as otherwise noted, that binding is stronger with certain specific moieties than it is to other moieties in general, that it is stronger than non-specific binding that may occur with a wide variety of moieties, and that binding is selective for certain moieties and does not occur to as strong an extent with others. In the extreme case of specific binding, very strong binding occurs with a single type of moiety, and there is no non-specific binding with any other moiety.

“Co-administer” means an administration of two, or more agents in conjunction with one another, including simultaneous and/or sequential administration.

“Cognate(s)” herein means complementary, fitting together, matching, such as, for instance, two jigsaw puzzles that fit one another, the cylinder mechanism of a lock and the key that opens it, the substrate binding site of an enzyme and the substrate of the enzyme, and a target and target binding protein that binds specifically thereto.

“Cognate binding moieties” herein means binding moieties that bind specifically to one another. Typically, but not always, it means a pair of binding moieties that bind specifically to one another. The moieties responsible for highly selective binding of a specific ligand and ligand receptor provide an illustrative example of cognate binding moieties. Another example is provided by the moieties that binds an antigen and an antibody.

“Composition” means any composition of matter comprising one or more constituents, such as a formulation.

“Comprised of” is a synonym of “comprising” (see below).

“Comprising” means including, without further qualification, limitation, or exclusion as to what else may or may not be included. For example, “a composition comprising x and y” means any composition that contains x and y, no matter what else it may contain. Likewise, “a method comprising x” is any method in which x is carried out, no matter what else may occur.

“Concentration” is used herein in accordance with its well-known meaning in the art to mean the amount of an item in a given amount of a mixture containing the item, typically expressed as a ratio. For example, 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 units of solvent volume; (B) Weight Percent (ii)=weight of solute per 100 units of total weight; (C) Weight Percent (iii) weight of solute per 100 units of solvent by weight; (D) Mass Percent=mass of solute per 100 mass units of solution; (E) Mole Fraction=moles of solute per total moles of all components; (F) Molarity=moles of solute per liter of solution (i.e., solute plus solvent); (G) Molality=moles of solute per Kg of solvent; and (H) Volume Molality=moles of solute per liter of solvent.

“Control region(s)” is used herein in accordance with its well-known meaning in the art, and except as noted otherwise, refers to regions in DNA or proteins that are responsible for controlling one or more functions or activities thereof. For instance, “expression control region” with reference to the control of gene expression, means the regions in DNA that are required for transcription to occur properly and that are involved in regulating when transcription occurs, how efficiently it occurs, when it is stopped, and the like.

“De novo” is used herein in accordance with its well-known meaning in the art, to denote something made from new. For instance, a de novo amino acid sequence is one not derived from a naturally occurring amino acid sequence, although, such a de novo sequence may have similarities with a naturally occurring sequence. De novo amino acid sequences can be generated, for instance, by a priori design, by combinatorial methods, by selection methods. They can be made, for example, by chemical synthesis, by semi-synthesis, and by a variety of recombinant DNA techniques, all of which are well know to those skilled in the art.

“Deleterious” means, as used herein, harmful. By way of illustration, “deleterious” processes include, for example, harmful effects of disease processes and harmful side effects of treatments.

“Derivative(s)” is used herein to mean derived from, in substance, form, or design, such as, for instance, a polypeptide that is based on but differs from a reference polypeptide, for instance, by alterations to its amino acid sequence, by fusion to another polypeptide, or by covalent modification.

“Disease(s)” a pathology, a condition that deleteriously affects health of a subject.

“Disorder(s)” a malediction, a condition that deleteriously alters health.

“Dysfunction” means, as used herein, a disorder, disease, or deleterious effect of an otherwise normal process.

“Effective amount” generally means an amount which provides the desired local or systemic effect. For example, an effective amount is an amount sufficient to effectuate a beneficial or desired clinical result. The effective amount can be provided all at once in a single administration or in fractional amounts that provide the effective amount in several administrations. The precise determination of what would be considered an effective amount may be based on factors individual to each subject, including their size, age, injury, and/or disease or injury being treated, and amount of time since the injury occurred or the disease began. One skilled in the art will be able to determine the effective amount for a given subject based on these considerations which are routine in the art. As used herein, “effective dose” means the same as “effective amount.”

“Effective route” generally means a route which 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 at the desired site of action an amount of the agent sufficient to effectuate a beneficial or desired clinical result.

“Endogenous” (such as endogenous gene) is used herein to refer to, for instance, genes and other aspects of DNA, such as control regions, that naturally occur in a genome and organism, unless otherwise indicated.

“Exogenous” (such as exogenous gene), unless otherwise indicated, is used herein generally to mean, for instance, DNA from an outside source, such as DNA introduced to a cell and incorporated into its genome.

“FBS” means fetal bovine serum.

“Formulation(s)” means a combination of at least one active ingredient with one or more other ingredients for one or more particular uses, such as storage, further processing, sale, and/or administration to a subject, such as, for example, administration to a subject of a specific agent in a specific amount, by a specific route, to treat a specific disease.

“Fragment(s)” herein means part of a larger entity, such as a part of a protein; for instance, a polypeptide consisting of less than the entire amino acid sequence of a larger polypeptide. As used herein, the term includes fragments formed by terminal deletion and fragments formed by internal deletion, including those in which two or more non-contiguous portions of a polypeptide are joined together to form a smaller polypeptide, which is a fragment of the original.

“Fusion protein(s)” herein means a protein formed by fusing all or part of two polypeptides, which may be either the same or different. Typical fusion proteins are made by recombinant DNA techniques, by end to end joining of nucleotides encoding the two (or more) polypeptides.

“Genetically engineered” herein means produced using a deliberate process of genetic alteration, such as by recombinant DNA technology, classical methods of genetic manipulation, chemical methods, a combination of all three, or other methods.

“Homolog(s)” herein means having homology to another entity, such as a protein that is homologous to another protein. Homologous means resembling in structure or in function.

“Ionization” herein means the change of net charge on a substance by at least one, including loss or gain of charge, such as the ionization of acetic acid in low pH solution, from HOAc to OAc− and H+.

“k” herein denotes an equilibrium co-efficient, in accordance with its standard meaning in chemistry.

“ka” herein denotes the dissociation constant of a particular hydrogen of a molecule, in accordance with its standard meaning in chemistry, such as, for example, the dissociation constant of the acidic hydrogen of acetic acid.

“kd” herein denotes a dissociation constant of a pair of chemical entities (or moieties), in accordance with its standard meaning in chemistry.

“Kit” means a collection of items used together for a given purpose or purposes.

“Ligand(s)” herein means a molecular entity that binds selectively and stoichiometrically to one or more specific sites on one more other molecular entities. Binding typically is non-covalent, but can be covalent as well. Avery few examples, among many others, are (a) antigens, which typically bind non-covalently to the binding sites on cognate antibodies; (b) hormones, which typically bind hormone receptors, non-covalently; (c) lectins, which bind specific sugars, non-covalently; (d) biotins, which bind multiple sites on avidin and other avidin-like proteins, non-covalently; (e) hormone antagonists, which bind hormone receptors and inhibit their activity and/or that of the corresponding hormone; and (f) hormone agonists, which similarly bind hormone receptors but stimulate their activity.

“Ligand-binding moiety(s)” herein means a molecular entity that binds a ligand, typically, a part of a larger molecular entity that binds the ligand, or a molecular entity derived therefrom.

“Ligand-binding protein(s)” herein means a protein that binds a ligand.

“Ligand moiety(s)” herein means a molecular entity that binds to a ligand-binding molecular entity in much the same way as does the corresponding ligand. A ligand moiety can be all of a ligand, or part of it, derived from a ligand, or generated de novo. Typically, however, the ligand moiety is more or less exclusively the aspect thereof that binds corresponding ligand-binding entities. The ligand moiety need not comprise, and the term generally does not denote, structural features other than those required for ligand binding.

“mEq” herein means milliequivalent(s).

“μEq” herein means microequivalent(s).

“Mimetic(s)” herein means a chemical entity with structural or functional characteristics of another, generally unrelated chemical entity. For instance, one kind of hormone mimetic is a non-peptide organic molecule that binds to the corresponding receptor in the same way as the corresponding hormone.

“mM” means millimolar; 10−3 moles per liter.

“Modified protein(s),” “modified polypeptide(s),” or “modified fragment(s)” herein means a protein or a polypeptide or a fragment of a protein or polypeptide comprising a chemical moiety (structure) other than those of the twenty naturally occurring amino acids that form naturally occurring proteins. Modifications most often are covalently attached, but can also be attached non-covalently to a protein or other polypeptide, such as a fragment of a protein.

“Moiety(s)” herein means a molecular entity that embodies a specific structure and/or function, without extraneous components. For instance, in most cases, only a small part of a ligand-binding protein is responsible for ligand binding. This part of the protein, whether continuously encoded or discontinuously, is an example of a ligand-binding moiety.

“Naturally occurring” means occurs in nature, without human intervention.

“Non-naturally occurring” means does not occur in nature or, if it occurs in nature, is not in its naturally occurring state, environment, circumstances, or the like.

“PBS” means phosphate buffered saline.

“Peptibody” refers to a molecule comprising an antibody Fe domain (i.e., CH2 and CH3 antibody domains) that excludes antibody CH1, CL, VH, and VL domains as well as Fab and F(ab)2, wherein the Fc domain is attached to one or more peptides, preferably a pharmacologically active peptide, particularly preferably a randomly generated pharmacologically active peptide. The production of peptibodies is generally described in PCT publication WO00/24782, published May 4, 2000, which is herein incorporated by reference in its entirety, particularly as to the structure, synthesis, properties, and uses of peptibodies.

“Peptide(s)” herein means the same as polypeptide; often, but not necessarily, it is used in reference to a relatively short polypeptide,

“pH” is used in accordance with its well-known and universal definition as follows:

pH=−log [H3O+].

“Pharmaceutical” as used herein means is acceptable for use in a human or non-human subject for the treatment thereof, particularly for use in humans, and approved therefor by a regulatory authority empowered to regulate the use thereof such as, for example, the Food and Drug Administration in the United States, European Agency for the Evaluation of Medicinal Products, Japan\'s Ministry of Health, Labor and Welfare, or other regulatory agency such as those listed in R. Ng, DRUGS: FROM DISCOVERY TO APPROVAL, Wiley-Liss (Hoboken, N.J.) (2004), which is herein incorporated by reference in its entirety, particularly as to regulatory authorities concerned with drug approval, especially as listed in Chapter 7. As used herein the phrase “wherein the composition has been approved for pharmaceutical use by an authority legally empowered to grant such approval” means an entity or institution or the like, established by law and by law charged with the responsibility and power to regulate and approve the use of drugs for use in humans, and in some cases, in non-humans. Approval by any one such agency anywhere meets this qualification. It is not necessary for the approving agency to be that of the state in witch, for instance, infringement is occurring. Example of such entities include the U.S Food and Drug Administration and the other agencies listed herein above.

As used herein, “pharmaceutical” also may refer to a product produced in accordance with good manufacturing practices, such as those described in, among others, Chapter 9 and Chapter 10, of R. Ng, DRUGS: FROM DISCOVERY TO APPROVAL, Wiley-Liss (Hoboken, N.J.) (2004), which is herein incorporated by reference in its entirety, particularly in parts pertinent to good manufacturing practices for pharmaceutical protein formulations, in particular, as set forth in Chapters 9 and 10.

“Pharmaceutically acceptable” is used herein in accordance with its well-known meaning in the art to denote that which is acceptable for medical or veterinary use, preferably for medical use in humans, particularly approved for such use by the US Food and Drug Administration or other authority as described above regarding the meaning of “pharmaceutical.”

“Polypeptide(s)” see “Protein(s).”

“Precursor(s)” is used herein in accordance with its well-known meaning in the art to denote an entity from which another entity is derived. For instance, a precursor protein is a protein that undergoes processing, such as proteolytic cleavage or modification, thereby giving rise to another precursor protein (which will undergo further processing) or a mature protein.

“Protein(s)” herein means a polypeptide or a complex of polypeptides, in accordance with its well-known meaning in the art. As used herein, “protein(s)” includes both straight chain and branched polypeptides. It includes unmodified and modified polypeptides, including naturally occurring modifications and those that do not occur naturally. Such modifications include chemical modifications of the termini, the peptide backbone, and the amino acid side chains; amino acid substitutions, deletions and additions; and incorporation of unusual amino acids and other moieties, to name just a few such modifications. It also includes “engineered” polypeptides and complexes thereof, such as, but not limited to, any polypeptide or complex of polypeptides that has been deliberatively altered in its structure by, for instance, recombinant DNA techniques, chemical synthesis, and/or covalent modification, including deliberate alteration of amino acid sequence and/or posttranslational modifications.

“Protonation” means the addition of at least one hydrogen.

“Self-buffering” means the capacity of a substance, such as a pharmaceutical protein, to resist change in pH sufficient for a given application, in the absence of other buffers.

“Semi-de novo” herein means (a) partly designed in accordance with a particular reference and or produced from a precursor, and (b) partly designed without reference to a particular reference (such as designed solely by general principles and not based on any particular reference). For example, a polypeptide made by producing a first peptide in a bacterial expression system, producing a second peptide by chemical synthesis, and then joining the two peptides together to form the polypeptide.

“Semi-synthesis” means as used herein a combination of chemical and non-chemical methods of synthesis.

“Subject” means a vertebrate, such as a mammal, such as a human. Mammals include, but are not limited to, humans, farm animals, sport animals, and pets. Subjects in need of treatment by methods and/or compositions of the present invention include those suffering from a disorder, dysfunction, or disease, or a side effect thereof, or from a side effect of a treatment thereof.

“Substantially” is used herein in accordance with its plain and ordinary definition to mean to a great extent or degree. For example, substantially complete means complete to a great extent, complete to a great degree. By way of further illustration, substantially free of residue means to a great extent free of residue, free of residue to a great degree. Should numerical accuracy be required, depending on context, “substantially,” as used herein means, at least, 80% or more, particularly 90% or more, very particularly 95% or more.

“Therapeutically effective” is used herein in accordance with its well-known meaning in the art to denote that which achieves an improvement in the prognosis or condition of a subject or that otherwise achieves a therapeutic objective, including, for instance, a reduction in the rate of progress of a disease even if a subject\'s condition, nonetheless, continues to deteriorate.

“Therapeutically effective amount” generally is used to qualify the amount of an agent to encompass those amounts that achieve an improvement in disorder severity. For example, effective neoplastic therapeutic agents prolong the survivability of the subject, inhibit the rapidly-proliferating cell growth associated with the neoplasm, or effect a regression of the neoplasm. Treatments that are therapeutically effective within the meaning of the term as used herein, include treatments that improve a subject\'s quality of life even if they do not improve the disease outcome per se.

“Treat,” “treating,” or “treatment” are used broadly in relation to the invention and each such term encompasses, among others, preventing, ameliorating, inhibiting, or curing a deficiency, dysfunction, disease, or other deleterious process, including those that interfere with and/or result from a therapy.

“Variant(s)” herein means a naturally occurring or synthetic version of, for instance, a protein that is structurally different from the original but related in structure and/or function, such as an allelic variant, a paralog, or a homolog of a protein.

The invention provides for the first time self-buffering protein formulations, particularly biopharmaceutical protein formulations, methods for making the formulations, and methods for using the formulations, among other things. Any protein that provides sufficient buffer capacity within the required pH range at a concentration suitable for its intended use can be prepared as a self-buffering protein formulation in accordance with the invention. The invention can be practiced with a variety of proteins, including both naturally-occurring proteins and “engineered” proteins, particularly biopharmaceutical proteins, as discussed further below.

The utility of proteins, particularly biopharmaceutical proteins, to be formulated in self-buffering compositions, particularly pharmaceutically acceptable compositions, has not been recognized prior to the invention herein disclosed. The influence of proteins in the regulation of physiological pH has been recognized and studied for some time. However, it has not heretofore been recognized that proteins, particularly biopharmaceutical proteins, can have enough buffer capacity to maintain a formulation within a desired pH range, without additional buffering agents.

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

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

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 the protein within 0.5 pH units of pH 6.3 is determined substantially by its histidine content. On this basis, the upper limit of the self-buffering capacity of the formulation is determined by the effective concentration of the histidine residues, 0.15 mM. The molar concentration of aspartic acid and glutamic acid in the formation is 0.9 mM. The total molar concentration of all three amino acids together, thus, is just a little over 1 mM, at the concentration of the formulation.

Humulin® is formulated at 3.5 g/ml. It has a molecular weight of about 6,000 Daltons and contains 2 aspartic acids, 8 glutamic acids, and 2 histidines. None of these amino acids is a particularly effective buffer at the pH of the formulation: 7.0 to 7.8. At this concentration the molar concentration of histidines, which are closest in pKa to the pH of the formulation, is 1.16 mM.

The biopharmaceutical lyophilates are reconstituted prior to use forming solutions or suspensions. Most of the lyophilates contain conventional buffers that maintain the proper pH of the reconstituted formulations. A few others, in which the protein concentration is low or the pH must be low (less than 3) or high (greater than 9.5), are, effectively unbuffered.

Thus, buffering is achieved in current biopharmaceutical protein formulations using conventional buffering agents. The ability of proteins by themselves to buffer pharmaceutical protein formulations has not been fully appreciated and has not been used for the manufacture of protein pharmaceuticals.

The determination of protein buffer capacity, typically, is important to developing self-buffering protein formulations in accordance with the invention. Pertinent thereto, methods for measuring buffer capacity and for determining the buffer capacity of proteins are described below. To allow ready comparability of data, protein buffer capacity must be expressed in comparable units and/or related to a buffer standard. Accordingly, the following section describes pH metrics and standards that meet these requirements, in accordance with the invention.

A widely accepted definition of buffering is the resistance to change in pH of a composition upon addition of acid or base. Buffer capacity thus often is defined as the ability of a composition to resist pH change.

Typically buffer capacity is expressed in terms of the amount of strong acid or base required to change the pH of a composition a given amount. Van Slyke provided the most widely used quantitative measure of buffer capacity, according to which, for a solution, buffer capacity is expressed as the amount of strong acid or base required to change the pH of one liter of the solution by one pH unit under standard conditions of temperature and pressure.

According to this measure, for instance, the buffer capacity of 1 liter of 5 mM HOAc, 5 mM NaOAc, pH 4.76 in pure water is 4.09×10−3 moles of a univalent strong base (i.e., 4.09×10−3 equivalents of base), which can be calculated as follows.

The Henderson-Hasselbalch equation for the solution is:

pH=log {[5 mM]NaOAc/[5 mM]HOAc}+4.76

Accordingly, the concentration, X, of a univalent strong base required to increase the pH of this buffer is:

4.76 to 5.76 is 5.76=log {[5 mM+X mM]NaOAc/[5 mM−X mM]HOAc}+4.76

Thus:

1.00=log {[5 mM+X mM]NaOAc/[5 mM−X mM]HOAc}

10.0=[5 mM+X mM]NaOAc/[5 mM−X mM]HOAc

10.0 (5 mM+X mM)/(5 mM−X mM)

50 mM−10X mM=5 mM+X mM

11X in M=45 mM

X=4.09 mM,

which, for one liter yields:

(4.09×10−3 moles/liter)(1 liter)(1 equivalent/mole)=4.09×10−3 equivalents

Thus, according to this measure, the buffer capacity of 1 liter of a 10 mM acetate buffer containing 5 mM NaOAc and 5 mM HOAC at a pH of 4.76 in pure water is 4.09×10−3 equivalents of base per liter per pH unit. Put other ways, the buffer capacity of the solution is 4.09 milliequivalents of base per liter per pH unit, 4.09 microequivalents of base per milliliter per pH unit, 0.409 microequivalents of base per 100 microliters per pH unit, 40.9 nanomoles of base per 10 microliters per pH unit, and 4.09 nanonmoles of base per microliter per pH unit.

The same calculation yields the following buffer capacity for other concentrations of this acetate buffer at pH 4.76. A 2 mM acetate buffer as above has a buffer capacity of 0.818 mEq per liter per pH unit. At 4 mM the buffer capacity is 1.636 mEq per liter per pH unit. The capacity at 5 mM is 2.045 mEq per liter per pH unit. At 7.5 mM the capacity is 3.068 mEq per liter per pH unit. At 10 mM the acetate buffer has a buffer capacity of 4.091 mEq per liter per pH unit. At 15 mM its capacity is 6.136 mEq per liter per pH unit.

It is worth noting that an acetate buffer solution at the pKa of acetic acid (pH 4.76) is equimolar in acetic acid and acetate base. (i.e., at the pKa the acid and base are present in equal amounts). As a result, the resistance to change in pH (buffer capacity) of an acetate buffer at the pKa of acetic acid is the same for addition of acid and base. The equipoise to acid and base is a general characteristic of buffering agents in buffers at a pH equal to their pKa.

At any other pH a buffer will contain different amounts of acid and base forms and, therefore, its resistance to change (i.e., its buffer capacity) upon addition of acid will not be the same as its resistance to change upon addition of base. As a result, it is preferable to define the capacity of such buffers 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.

The partitioning in a buffer between acid and base forms in a given composition, such as a pH standard, can be calculated at any pH and buffer concentration using the procedures set forth above in describing the buffer capacity of 10 mM NaOAc at pH 4.76 plus or minus (containing equimolar amounts of acetic acid and sodium acetate). And the results can be used to define the buffer capacity of a standard for reference use.

Thus, for instance, the partitioning of acetic acid into acetic acid and acetate base in a solution at pH 5.0 can be calculated readily using the foregoing procedures, and from this the buffer capacity can be calculated for both base and for acid addition. Calculated this way, the theoretical buffer capacity of 10 mM sodium acetate buffer over the range from pH 5.0 to 5.5 is approximately 2.1 mM per 0.5 pH unit and 4.2 mM per pH unit. Put another way, the buffer capacity of the buffer, theoretically, is approximately 4.2 μEq per ml of buffer solution per unit of pH change. Similarly, the theoretical buffer capacity of 10 mM sodium acetate buffer over the range from pH 5.0 to 4.0 is 4.9 mM, and, put another way, 4.9 μEq per ml of buffer per unit of pH change over a given range of pH.

While such calculations often are quite useful in many cases, empirical standards and empirical determinations are preferred. Among particularly preferred empirical standards are sodium acetate buffers over the range of pH 5.0 to 4.0 and pH 5.0 to 5.5 as exemplified in Examples 1 and 2. Especially preferred are sodium acetate buffers in accordance therewith in which the total acetate concentration is, in particular, 10 mM, preferably 5 mM, especially 4 mM, among others as set forth elsewhere herein.

Acetate buffers at pH 5.0 are more resistant to change in pH upon addition of acid than upon addition of base, as discussed above. In a preferred empirical standard of buffer capacity, the buffer capacity of a standard acetate buffer such as these is defined as: (i) the slope of the least squares regression line calculated for base titration data for the buffer from pH 5.0 to pH 5.5, and (ii) the slope of the least squares regression line calculated for acid titration data for the buffer from pH 5.0 to pH 4.0. The preparation of standard acetate buffers and the determination of their buffer capacities are described in Examples 1, 2, and 3. It is to be appreciated that much the same methods can be used to establish and use buffer capacity standards using other suitable buffering agents.

In measuring the buffer capacity of a self-buffering protein composition in accordance with the invention, it often is convenient to express the buffer capacity in terms of the concentration of a standard buffer at the same pH having the same buffer capacity. When a standard is used that is not at the pKa of the buffering agent, such as a sodium acetate buffer initially at pH 5.0, in accordance with the invention the self-buffering composition is defined as having a buffer capacity equal to or greater than that of the standard, if either its buffer capacity upon base titration or its buffer capacity upon acid titration (or both) is equal to or exceeds the corresponding buffer capacity of the standard.

It is to be further appreciated that the pH of self-buffering protein compositions in accordance with the invention generally will not be at the pKa of the self-buffering protein, or any acid-base substituent therein. Indeed proteins are polyprotic and, as discussed herein, often will have several substituents, each with a somewhat different pKa that contribute to its buffer capacity in a given pH range. Accordingly, the buffer capacity of self-buffering protein formulations in accordance with the invention preferably is determined empirically by both acid titration and base titration over a given range of pH change from the desired pH of the composition. In preferred embodiments in this regard, the buffer capacity is determined by titrating with acid and separately with base over a change of respectively + and −1 pH unit from the starting pH of the formulation. In particularly preferred embodiments, the titration data is collected for a change in pH of plus or minus 0.5 pH units. As described in the Examples, the buffer capacity is the slope of the least squares regression line for the data for pH as a function of equivalents of acid or base added to the composition over the range of titration.

a. Empirical Measures and Standards of Buffer Capacity

In certain preferred embodiments of the invention, the measure of buffer capacity is an empirical standard. Among preferred empirical standards in this regard are a particular volume of an aqueous solution at a particular temperature and a particular pH, containing a particular buffering agent at a particular concentration and either no other components than water, or one or more other particular components, each at a particular concentration.

A particularly preferred specific standard for determining buffer capacity in accordance with various aspects and preferred embodiments of the invention is 10 mM sodium acetate pH 5.00 in pure water free of other constituents at 21° C. in equilibrium with ambient air at 1 atmosphere, as described in Examples 1 and 2, preferably expressed in equivalents per unit volume per pH unit, such as μEq/ml-pH. Buffer capacity of the standard should be measured empirically as described in Examples 1, 2, and 3, and as further discussed elsewhere herein.

A particularly preferred specific standard for determining buffer capacity in accordance with various aspects and preferred embodiments of the invention is 10 mM sodium acetate pH 4.76 in pure water free of other constituents at 21° C. in equilibrium with ambient air at 1 atmosphere, as described in Examples 1 and 2, preferably expressed in equivalents per unit volume per pH unit, such as μEq/ml-pH. Buffer capacity of the standard should be measured empirically as described in Examples 1, 2, and 3, and as further discussed elsewhere herein. According to the Henderson-Hasselbalch equation, as noted above, the calculated buffer capacity of this standard over the range of pH 4.76 plus or minus 1 pH unit is 4.09 microequivalents per milliliter per pH unit (4.09 μEq/ml-pH).

A variety of other buffers are available for use as standards in other ranges of pH in accordance with various aspects and preferred embodiments of the invention in this regard. Reference buffers are particularly preferred in this regard, such as those well-known and routinely employed for analytical chemistry determinations. A variety of such buffering agents are set forth in textbooks on analytical chemistry and in monographs on the accurate determination of pH and buffer capacity.

Also useful in the invention in this regard are biological buffers, such as those described in, among other texts: TEITZ TEXTBOOK OF CLINICAL CHEMISTRY, 3rd Ed., Burtis and Ashwood, eds., W.B. Saunders Company, Philadelphia, Pa. (1999), in particular in Tables 50-13 to 50-16, which are herein incorporated by reference in their entireties as to buffering agents and buffers and their use as pH and/or buffer capacity standards in accordance with the invention in this respect; THE TOOLS OF BIOCHEMISTRY, Terrance G. Cooper, John Wiley & Sons, New York, N.Y. (1977), in particular Chapter 1, pages 1-35, which is herein incorporated by reference in its entirety as to buffering agents and buffers and their use as pH and buffer capacity standards in accordance with the invention in this respect, most particularly as to Tables 1-3, 1-4, and 1-5 and text relating thereto, and PROTEIN PURIFICATION PRINCIPLES AND PRACTICE, 3rd Ed., Robert K. Scopes, Springer-Verlag, New York, N.Y. (1994), in particular pages 160-164, especially therein Tables 6.4 and 6.5 and text relating thereto, Chapter 12, section 3, pages 324-333, especially therein Tables 12-4 and 12-5 and text relating thereto, and all of Appendix C: Buffers for Use in Protein Chemistry, which are herein incorporated by reference in their entirety as to buffering agents and buffers and their use in accordance with the invention in this respect.

Since some dissolved gases in water react with OFF and/or H3O+, however, the empirically determined buffer capacity of the standard solution may vary somewhat from the theoretical value. Hence, the definition of the standard requires that the solution be in equilibrium with the atmosphere at a pressure of 1 atmosphere. In addition, the buffer standard must be held in and contacted only with materials that do not alter its components or its buffer capacity, such as those that leach acids, bases, or other reactants that may alter the effective concentration or activity of the acetate buffer in any way that would alter its buffer capacity. Given both of the foregoing, atmospheric equilibration and inertness of the container, buffer capacity of the standard will scale directly and linearly with its volume. Accordingly, the buffer capacity of 100 ml will be 1/10 that of 1.00 liter, and the buffer capacity of 10 ml will be 1/100 that of 1.00 liter. Accordingly, the volume of the standard can be adjusted for convenience and then normalized back to 1 liter as desired.

It may not always be convenient to make the foregoing 10 mM acetate buffer capacity standard for field use. However, a variety of other buffer capacity standards can be made and used in the same way as the acetate buffer, using a variety of other buffering agents. Provided only that the buffering standards are prepared properly, they can be calibrated against the acetate buffering standard described above and then used in the field. The results obtained with such alternative standards may then be expressed in terms of the foregoing acetate standard without substantial distortion or error.

The buffer capacity of such alternative standards also can be calibrated by calculation. To do so, the buffer capacity of the alternative standard is determined directly and expressed in mEq per unit volume per unit of pH. Determinations based on the alternative standard then can be normalized to the acetate standard using the ratio between the buffering capacities expressed in mEq per unit volume per unit of pH of the alternative and the acetate standards.

Using such methods, which are commonly employed in metrology to relate field standards back to a reference standard, the acetate buffer standard described above provides a portable, scalable, reliable, and accurate reference for determining the buffer capacity of any composition that readily can be compared with disparate measures made on other compositions using similar methods.

b. Preparation of Buffer Capacity Standards

Buffer capacity standards can be prepared using well-established methods of analytical chemistry. See for instance, ANALYTICAL CHEMISTRY, 3rd Ed., Douglas A. Skoog and Donald M. West, Holt, Rinehart and Winston, New York (1979), particularly chapter 9 (pages 186-226), chapter 10 (pages 227-233), and methods described on pages 583-588; TEITZ TEXTBOOK OF CLINICAL CHEMISTRY, 3rd Ed., Burtis and Ashwood, eds., W.B. Saunders Company, Philadelphia, Pa. (1999), in particular Chapter 1 regarding general laboratory techniques for preparing and calibrating buffers and Tables 50-13 to 50-16; THE TOOLS OF BIOCHEMISTRY, Terrance G. Cooper, John Wiley & Sons, New York, N.Y. (1977), in particular Chapter 1, pages 1-35, and Tables 1-3, 1-4, and 1-5 and text relating thereto; PROTEIN PURIFICATION PRINCIPLES AND PRACTICE, 3rd Ed., Robert K. Scopes, Springer-Verlag, New York, N.Y. (1994), in particular pages 160-164, especially therein Tables 6.4 and 6.5 and text relating thereto, Chapter 12, section 3, pages 324-333, especially therein Tables 12-4 and 12-5 and text relating thereto, and all of Appendix C: Buffers for Use in Protein Chemistry; and REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY, 21st Ed., Beringer et al. Editors, Lippincott, Williams & Wilkins, Philadelphia, Pa. (2005), particularly in parts relating to buffering agents, buffers, buffer capacity and the like; each of which is herein incorporated by reference in its entirety particularly as to the preparation and use of buffers and buffer capacity standards in accordance with the invention in this respect.

The water used for preparing buffer capacity standards should be highly purified, preferably Type I water, such as milliQ water, or triple distilled water. The buffer reagents should be pure and, in particular, free of any substance that can alter the pH or buffer capacity of the standard solution, such as Reference Grade or ACS Reagent Grade reagents suitable for use in demanding analytic chemical analyses, as described in the foregoing references, TEITZ and REMINGTON cited above in particular, which are hereby incorporated by reference in their entireties particularly in parts pertinent to analytical grade water and reagents.

The exact compositions of the buffer reagents must be well established. The molecular weight of the buffer reagents must be known accurately for each buffer reagent. The molecular weights must be for the exact reagent that will be used and must include the weight of adducts such as hydrates that are present in the reagent. The effective number of hydrogen donors or hydrogen acceptors per molecule must be known accurately for each buffer reagent. The proportional distribution of different forms, such as hydrates, must be known for each reagent that contains a mixture of such forms. Concentrations of liquid buffer reagents much be known exactly, preferably in moles/volume and in moles/mass (e.g., moles/liter and moles/gm or kg. Hygroscopic agents must be dried to remove moisture so that reagent can be accurately weighed.

Generally speaking, the information provided by well-established vendors of reagents and reference grade chemicals is sufficiently accurate for the preparation of buffer capacity standards as described above. And well-known standard techniques routinely employed in analytical chemistry can be used to dry “hygroscopic reagents” so that they can be weighed accurately.

As described therein, well established and routinely employed analytical chemistry methods can be employed to prepare and calibrate acid and base solutions, such as 1 N HCl and 1 N NaOH (to name just two) for titrating buffer capacity standard solutions, as well as sample protein solutions, to determine buffer capacity. It should be noted that the preparation of NaOH solutions for titration should be done so as to eliminate inaccuracies that arise from the interaction of certain dissolved gases with basic solutions, and the pH altering effects of their solvation. See for instance Skoog and West (1979) and other references cited above regarding the preparation and calibration of buffers and buffer standards, which are herein incorporated by reference in their entireties particularly in parts pertinent to the preparation of standard solutions for titration, as discussed above.

c. Empirical Measurement of Buffer Capacity

Titration of standards and samples to determine buffer capacity can be done using well-known, routine methods. Titrations can be carried out manually. They also can be carried out using an autotitrator. A wide variety of autotitrators that are suitable for use in the invention in this regard are commercially available from numerous vendors. Methods suitable for use in the invention in this regard are the same as those described in the references cited above regarding preparation and calibration of buffer standards, each of which is incorporated herein by reference in its entirety particularly in parts pertinent to the titration of known and unknown solutions to determine their buffer capacity.

a. Determination of Protein Hydrogen Equilibria and Buffer Capacity

Proteins invariably contain many acidic and basic constituents. As a result hydrogen ion equilibrium of proteins is highly complex. In fact, a complete description of the hydrogen ion equilibria of a protein in a given environment is beyond the reach of current theoretical and computational methods. Empirical measurements of protein buffer capacities, thus are preferred. Methods developed for precise empirical measurement of protein hydrogen equilibria, which can be and are routinely employed by those skilled in the art, are well-suited to measuring the buffering properties of proteins pertinent to the development of self-buffering protein formulations in accordance with the invention. Thus, the pH titration curves of proteins can be determined in accordance with the invention by well-known methods such as those described in and exemplified by pH titration studies of Tanford and co-workers on ribonuclease. See C. Tanford, “Hydrogen Ion Titration Curves of Proteins,” in T. Shedlovsky (ed.), ELECTROCHEMISTRY IN BIOLOGY AND MEDICINE, John Wiley and Sons, New York, 1955, Ch. 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, particularly pages 554-567, all of which are herein incorporated by reference particularly in parts pertinent to hydrogen ion titration of proteins and to the determination of buffering action and buffer capacity of proteins.

However, the present invention does not require such precise determinations as those described in the foregoing references. Rather, the buffering properties and buffer capacity of proteins in accordance with the invention can be determined using the methods described in standard references on analytical chemistry and biochemistry, such as, for instance, Skoog (1979), Cooper (1977), and Scopes (1994), cited above, each of which is herein incorporated by reference in its entirety particularly as to the empirical determination of titration curves, particularly of proteins within a given range of pH in accordance with the invention.

The determination of titration curves and buffer capacity in accordance with the invention is described in detail for numerous acetate buffers and a variety of pharmaceutical proteins in the Examples below. Thus, the pH titration curves of proteins can be determined empirically in accordance with such methods as described in the foregoing references over particular limited ranges of pH that are of interest to a given formulation. In many respects these methods are the same as those used in analytical chemistry for the titration of small molecules such as acetate buffers (as illustrated in the Examples). Somewhat greater care must be taken, however, in handling proteins to maintain the conformation and function required for effective formulation.

Protein titrations may be carried out manually or using automated titrators. Equipment for manual titration and automated titrators are readily available from a large number of suppliers and vendors. Methods suitable for determining pH titration curves and buffer capacity of proteins are exemplified in the Examples by reference to titration of acetate buffer standards and to titration of several different therapeutic proteins over defined ranges of pH. These methods can be employed to determine the hydrogen ionization behavior and buffer capacity of any other protein in accordance with the invention.

It is a particular aspect of the invention to determine the buffer capacity of proteins as a function of concentration in solution. In a preferred method in this regard, solutions of a given protein are prepared in a graded series of concentrations. A pH titration curve is determined for the protein at each concentration over the pH range of interest. Preferably titration curves are determined for the range of interest using both base titration and acid titration. The data are, in certain preferred embodiments, plotted on a graph of equivalents of acid or base added versus the measured pH of each solution. Typically, for ranges of about 0.5 to 1.0 pH unit, the titration data for each concentration closely fit a straight line, preferably determined by a least squares regression analysis. In preferred embodiments in this regard, buffer capacity for the protein at each concentration is equated to the slope of the regression line, expressed in units of equivalents per ml per pH unit (or fractions thereof). Also useful in the invention in this regard is the relationship between the buffer capacity of the protein and its concentration. In certain preferred embodiments, this relationship is determined by a least squares regression analysis of the best straight line fit of the buffer capacity data determined in accordance with the foregoing plotted on a graph of buffer capacity versus protein concentration.

Empirical data on the buffer capacity of proteins in accordance with the invention preferably is related to the buffer capacity of a standard acetate buffer. That is, in particularly preferred embodiments of the invention in this regard, the buffer capacity of a given protein at a given concentration in a given formulation, determined as above, is equated to the concentration of a standard acetate buffer having the same buffer capacity.

While empirical determinations as described herein are generally a crucial aspect of formulating self-buffering compositions in accordance with various aspects and preferred embodiments of the invention, theoretical and computational methods also can be productively employed to guide the design, manufacture, and use of such compositions (in conjunction with empirical determinations), as described below.

b. Prediction of Protein Hydrogen Ion Equilibria and Buffer Capacity

The ionization of hydrogen in proteins is complex but can be broken down in general terms into pH ranges defined by the ionizable hydrogens of amino acid side chains, and the terminal amino and carboxyl groups. The pKa of terminal carboxyls in polypeptides typically ranges around 3.1. The pKa of the acidic hydrogens in the side chains of aspartic acid and glutamic acid range around 4.4. The pKa of histidine in polypeptides ranges around 6.0. The terminal amino group hydrogen ionization pKa typically ranges around 7.5. The sulfhydryl in cysteine has a pKa range around 8.5. The tyrosine hydroxyl and the lysine amine both have pKas ranging around 10. The pKa of arginine ranges around 12.

Conformational folding typically partitions large polypeptides and proteins in polar solvents into exposed solvent-accessible regions and more or less non-polar core regions that have little or no contact with the ambient environment. Folding produces many environments between these two extremes. Furthermore, the micro environment around a given amino acid side chain in a protein typically is affected by one or more of: solvent effects; binding of ions; chelation; complexation; association with co-factors; and post-translational modifications; to name just a few possibilities. Each of these can influence the pKa of a given amino acid ionization in a protein. The pKas for specific residues in a given protein, thus, can vary dramatically from that of a free amino acid.

Indeed, the perturbation of pKas by microenvironments of amino acids in proteins has been used to study the folding of proteins and the disposition and charge state of specific amino acids in folded proteins. The protein titration curves reported by Tanford and others are complex with a few broad features in common. Typically only some of the ionizable protons are accounted for in the titration curves. Others apparently are located in the core and are inaccessible to solvent. The pKas of individual side chains of the same type that can be detected in some cases can be distinguished from one another. Nonetheless, while detectably different, their pKas generally are close to that of the free amino acid.

The strongest buffering action of proteins does not generally occur at the isoelectric point, as may be mistakenly supposed. In fact, buffering depends on the amino acid side chain hydrogens and the terminal hydrogens, and therefore occurs in ranges spanning the pKas of the ionizable hydrogens in the free amino acids, as discussed above. The most important of these, for formulating compositions of proteins, especially certain pharmaceutical proteins that are more soluble and/or more stable, among other things, at weakly acidic pH (pH 4 to 6), is buffering action that occurs in the range of the pKas of the carboxyl hydrogen of the amino acids aspartic acid and glutamic acid; that is, pH 4.0 to 5.5, particularly around 4.5.

There are a variety of ways available for estimating the buffer capacity of a given protein in a given solution at a given pH. Methods range from highly technical and complex computer models to those that can be carried out on a hand calculator. None of the methods is complete or entirely accurate; but, they can in some instances provide useful estimates.

For instance, a potentially useful idea of buffer capacity in some instances may be calculated for a protein in a solution based on its amino acid composition, the pKas (in the solvent in question) of the terminal amine and carboxy groups and the amino acid side hydrogen donors and acceptors, the concentration of the protein, and the pH of the solution.

For example, a potentially useful estimate of the buffer capacity of a protein at pH in the range of the pKa of the side chain carboxyl hydrogen of glutamic acid (as a free amino acid), can be gained from the molecular weight of the protein and the number of glutamic acid residues it contains. Dividing the former by the latter provides the weight per equivalent of glutamic acid and, therefore, the weight per equivalent of ionizable hydrogen at the pKa of glutamic acid. Since glutamic acid and aspartic acid side chain carboxyl groups have nearly the same pKas, results of such calculations for the two should be added together to yield an estimate of buffer capacity in a range around both their pKas. The estimated buffer capacity of a solution of the protein at the pKa can be calculated from the protein\'s concentration in the solution and the intrinsic factor just provided, namely weight per equivalent of ionizable hydrogen. Dividing the concentration by the weight per equivalent yields an estimate for the buffer capacity in units of Eq/volume. Such estimates often will be too high, since some residues usually are sequestered in regions of the protein not accessible to the solvent, and, therefore, do not contribute to its actual buffer capacity. It may be possible in certain instances to account for the effect of sequestering on buffer capacity. For instance, a fractional co-efficient that reflects theoretical or empirical estimates of sequestering can be applied to adjust the original calculation.

Such calculations generally will be of less utility and less accurate than empirical determinations of protein buffer capacity, in accordance with the methods described elsewhere herein. But they can be useful to provide rough maximum estimates of the buffer capacity of proteins in solution.

The invention herein disclosed may be practiced with any protein that provides sufficient buffer capacity in a desired pH range within the parameters of protein concentration and the like required for a desired formulation. Among preferred proteins in this regard are pharmaceutical proteins for veterinary and/or human therapeutic use, particularly proteins for human therapeutic use. Also among preferred proteins are proteins that are soluble in aqueous solutions, particularly those that are soluble at relatively high concentrations and those that are stable for long periods of time. Additionally, among preferred proteins are those that have a relatively high number of solvent accessible amino acids with side chain hydrogen ionization constants near the pH of the desired buffering action.