High concentration anti-sclerostin antibody formulations

0001The present invention relates to a high concentration antibody preparation. Cross-reference of related applications The present application claims the priority benefit of US Provisional Application No. 61 / 334,986 filed May 14, 2010, the disclosure of which is incorporated herein by reference in its entirety.

0002High-concentration liquid antibody formulations are useful for delivering smaller doses. However, high-concentration protein preparations pose some problems. One problem is instability due to the formation of fine particles. Another problem is the increase in viscosity as a result of many intermolecular interactions due to the polymeric nature of the antibody. Highly viscous formulations are difficult to manufacture, aspirate, and inject into syringes. The use of force in the handling of viscous formulations can result in excessive foaming, which can result in denaturation and inactivation of the active bioform.

0003U.S. Pat. Nos. 6,875,432 and U.S. Patent Application Publication Nos. 2006/0182740, 2007/0172479, and 2008/0160014 disclose antibody preparations and methods for making them. None of these publications disclose the antibodies referred to herein.

<p num="0004"><patcit num="1"><text>U.S. Pat. No. 6,875,432</text></patcit><patcit num="2"><text>U.S. Patent Application Publication No. 2006/0182740</text></patcit><patcit num="3"><text>U.S. Patent Application Publication No. 2007/0172479</text></patcit><patcit num="4"><text>U.S. Patent Application Publication No. 2008/0160014</text></patcit></p>

0005The present disclosure is based on the finding that the addition of low concentrations, eg, 5-10 mM calcium acetate, reduced the effective viscosity in formulations containing high concentrations of the selected anti-sclerostin antibody. In contrast, the same concentration of calcium acetate did not significantly reduce the viscosity of other antibody formulations. In one aspect, the formulation, in sterile, liquid or reconstituted liquid form, is (a) an anti-sclerostin antibody at a concentration of at least 70 mg / mL, SEQ ID NOs: 1-5 (Ab-A and Ab-1). CDR), 15 ~ 20 (Ab-B CDR), 25 ~ 30 (Ab-C CDR), 35 ~ 40 (Ab-D CDR), 45 ~ 50 (Ab-2 CDR), 55 ~ 60 (Ab-3) And Ab-15 CDR), 73-78 (Ab-4 and Ab-5 CDR), 91-96 (Ab-6 CDR), 101-106 (Ab-7 CDR), 111-116 (Ab-8 CDR) , 121-126 (Ab-9 CDR), 131-136 (Ab-10 CDR), 141-146 (Ab-11 and Ab-16 CDR), 159-164 (Ab-12 CDR), 169-174 (Ab) -13 and Ab-14 CDR), 187-192 (Ab-17 and Ab-18 CDR), 201-206 (Ab-19, Ab-20, and Ab-23 CDR), 225-229 (Ab-21 and Ab-22 CDR), Alternatively, an antibody and a calcium salt having a concentration in the range of about 1 mM to about 20 mM, or about 5 mM to about 10 mM, containing a set of 6 CDRs selected from the group consisting of 239 to 244 (Ab-24 CDR). This product has an absolute viscosity of about 10 cP or less. The absolute viscosities described herein are adjusted using a Brookfield LV-DVII cone plate viscometer with a CPE-40 spindle, where the temperature of the matching sample cup is adjusted by a circulating water tank at a constant 25 ° C. It was measured.

0006In some embodiments, the calcium salt is selected from the group consisting of calcium acetate, calcium carbonate, and calcium chloride. In one embodiment, the calcium salt is calcium acetate. Alternatively, in some embodiments, the calcium salt is at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17 compared to the same formulation of the calcium salt-free antibody. %, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% , 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, or more, at concentrations that reduce the viscosity of the antibody formulation.

0007In a related aspect, the formulation, in sterile, liquid or reconstituted liquid form, is (a) an anti-sclerostin antibody at a concentration of about 70 mg / mL to about 200 mg / mL, SEQ ID NOs: 1-5 (Ab-). A and Ab-1 CDR), 15 ~ 20 (Ab-B CDR), 25 ~ 30 (Ab-C CDR), 35 ~ 40 (Ab-D CDR), 45 ~ 50 (Ab-2 CDR), 55 ~ 60 (Ab-3 and Ab-15 CDRs), 73-78 (Ab-4 and Ab-5 CDRs), 91-96 (Ab-6 CDRs), 101-106 (Ab-7 CDRs), 111-116 ( Ab-8 CDR), 121-126 (Ab-9 CDR), 131-136 (Ab-10 CDR), 141-146 (Ab-11 and Ab-16 CDR), 159-164 (Ab-12 CDR), 169-174 (Ab-13 and Ab-14 CDRs), 187-192 (Ab-17 and Ab-18 CDRs), 201-206 (Ab-19, Ab-20, and Ab-23 CDRs), 225-229 (Ab-21 and Ab-22 CDR), or 239 ~ 244 (Ab-24) The preparation comprises an antibody comprising a set of 6 CDRs selected from the group consisting of CDRs) and (b) calcium acetate at a concentration in the range of about 5 mM to about 15 mM, or about 5 mM to about 10 mM. It has an absolute viscosity of about 10 cP or less. Alternatively, in some embodiments, calcium acetate is at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17 compared to the same formulation of the calcium acetate-free antibody. %, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50% , 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, or more, at concentrations that reduce the viscosity of the antibody formulation.

0008It also provides a method of reducing the viscosity of a protein formulation, which comprises adding calcium acetate at a concentration of about 1 mM to about 20 mM to an anti-sclerostin immunoglobulin formulation, which is about 70 mg / mL. The viscosity of a formulation containing immunoglobulin at a concentration of ~ about 200 mg / mL and containing calcium acetate is reduced compared to the viscosity of antibody formulations containing no calcium acetate.

0009In another aspect, the formulation is sterilized, has an absolute viscosity of about 10 cP or less, (a) Ab-5 at a concentration of at least 70 mg / mL to about 200 mg / mL, and (b) about 1 mM to about 20 mM. Concentrations in the range of 1% w / v to about 12% w / v, such as polyols such as sucrose. In certain embodiments, the amount of polyol is in the range of about 4% to 10%. In some embodiments, the immunoglobulin comprises the amino acid sequence of SEQ ID NO: 86 (Ab-5 heavy chain variable region) and / or SEQ ID NO: 84 (Ab-5 light chain variable region).

0010In another aspect, the formulation is sterilized, has an absolute viscosity of about 10 cP or less, (a) Ab-5 at a concentration of at least 70 mg / mL to about 200 mg / mL, and (b) about 1 mM to about 20 mM. Concentrations in the range of (c) and amounts in the range of about 4% w / v to about 6% w / v, such as polyols such as sucrose.

0011In any of the above embodiments, in some embodiments, the formulation further comprises (c) an acetate buffer having a concentration of about 5 mM to about 15 mM, or about 5 mM to about 10 mM, such as sodium acetate. .. In some embodiments, the total concentration of acetate is from about 10 mM to about 50 mM, or from about 20 mM to about 40 mM.

0012In a different embodiment, the formulation, in sterile, liquid or reconstituted liquid form, is (a) an anti-sclerostin antibody at a concentration of about 70 mg / mL to about 200 mg / mL, SEQ ID NOs: 1-5 (Ab-). A and Ab-1 CDR), 15 ~ 20 (Ab-B CDR), 25 ~ 30 (Ab-C CDR), 35 ~ 40 (Ab-D CDR), 45 ~ 50 (Ab-2 CDR), 55 ~ 60 (Ab-3 and Ab-15 CDRs), 73-78 (Ab-4 and Ab-5 CDRs), 91-96 (Ab-6 CDRs), 101-106 (Ab-7 CDRs), 111-116 ( Ab-8 CDR), 121-126 (Ab-9 CDR), 131-136 (Ab-10 CDR), 141-146 (Ab-11 and Ab-16 CDR), 159-164 (Ab-12 CDR), 169-174 (Ab-13 and Ab-14 CDRs), 187-192 (Ab-17 and Ab-18 CDRs), 201-206 (Ab-19, Ab-20, and Ab-23 CDRs), 225-229 (Ab-21 and Ab-22 CDR), or 239 ~ 244 (Ab-24) Concentrates in the range of (b) about 10 mM to about 50 mM acetate, or about 20 mM to about 40 mM acetate, and acetates and antibodies, including a set of 6 CDRs selected from the group consisting of CDR). / Or including acetate buffer, the formulation has an absolute viscosity of about 10 cP or less. In some embodiments, the acetate and / or buffer comprises calcium acetate and / or sodium acetate. Alternatively, in some embodiments, the acetate and / or buffer is at least 10%, 11%, 12%, 13%, as compared to the same formulation of the antibody that does not contain the acetate and / or buffer. 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30% , 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47 %, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, or more, the viscosity of the antibody preparation Exists at a reduced concentration.

0013In any of the aforementioned embodiments, in some embodiments, the total concentration of ions in solution (cations and anions) is from about 20 mM to about 70 mM, or from about 30 mM to about 60 mM. In any of these embodiments, the total osmolarity is less than about 400 mOsm / L or 350 mOsm / L, preferably close to isotonic, eg 250-350 mOsm / L. In some embodiments, the present formulation is hypotonic. For example, in such an embodiment, the osmolal concentration of the present formulation is less than about 250 mOsm / L. In another embodiment, the present formulation is hypertonic. Therefore, in such an embodiment, the total osmolal concentration of the present formulation is greater than about 350 mOsm / L.

0014In any of the formulations described herein, in some embodiments, the anti-sclerostin antibody in the formulation is the antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-2. , Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-15, Ab-16, Ab It can contain a mature heavy chain and / or light chain variable region of any of -17, Ab-19, Ab-21, Ab-23, or Ab-24. Thus, in certain embodiments, the antibody is SEQ ID NO: 14 (Ab-1 heavy chain variable region) and / or SEQ ID NO: 12 (Ab-1 light chain variable region), or SEQ ID NO: 68 (Ab-15 heavy chain). Variable region), and / or SEQ ID NO: 66 (Ab-15 light chain variable region), or SEQ ID NO: 86 (Ab-5 heavy chain variable region), and / or SEQ ID NO: 84 (Ab-5 light chain variable region), Or SEQ ID NO: 154 (Ab-16 heavy chain variable region) and / or SEQ ID NO: 152 (Ab-16 light chain variable region), or SEQ ID NO: 182 (Ab-14 heavy chain variable region) and / or SEQ ID NO: 180 ( Ab-14 light chain variable region), or SEQ ID NO: 208 (Ab-19 heavy chain variable region) and / or SEQ ID NO: 207 (Ab-19 light chain variable region), or SEQ ID NO: 216 (Ab-20 heavy chain variable region). ) And / or SEQ ID NO: 214 (Ab-20 light chain variable region), or SEQ ID NO: 220 (Ab-23 heavy chain variable region) and / or SEQ ID NO: 218 (Ab-23 light chain variable region), or SEQ ID NO: 238. Contains the amino acid sequence of (Ab-22 heavy chain variable region) and / or SEQ ID NO: 236 (Ab-22 light chain variable region). In some embodiments, the antibody is an antibody Ab-A, Ab-B, Ab-C, Ab-D, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7. , Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-15, Ab-16, Ab-19, Ab-23, or Ab-24. Includes and / or light chain variable region. Some embodiments

0015In any of the formulations described herein, in some embodiments, the anti-sclerostin antibody is Ab-4 or Ab-5, Ab-13 or Ab-14, or Ab-19, Ab-20. , Or a CDR of any of Ab-23, or a mature heavy chain and light chain variable region, or a mature heavy chain and light chain. In some embodiments, the antibody is 10 in any of the formulations described herein.^-7Or less K_DAnd binds to sclerostin of SEQ ID NO: 1 (lower numbers mean higher binding affinity).

0016In any of the formulations described herein, in some embodiments, the antibody in the formulation is present at a concentration of at least 120 mg / mL, or at least 140 mg / mL. In any of the formulations described herein, in some embodiments, the absolute viscosity of the formulation is about 8 cP or less, or about 6 cP or less. In an alternative embodiment, the antibody in the present formulation is present at a concentration of about 70 mg / mL to about 130 mg / mL, and the present formulation has an absolute viscosity of about 10 cP or less.

0017In some embodiments, any of the formulations described herein further comprises a polyol in an amount ranging from about 4% w / v to about 6%, such as sucrose. In some embodiments, the present formulation comprises about 9% sucrose. In some embodiments, any of the formulations described herein are optionally other pharmaceutically acceptable excipients such as salts, buffers, amino acids, stabilizers, polyols, etc. Includes isotonic agents, surfactants, fillers, antifreezes, rioprotectants, antioxidants, metal ions, chelating agents, and / or preservatives. In some embodiments, the present formulation has less than 0.05% by weight of surfactant.

0018In any of the formulations described herein, in some embodiments, the formulation has a pH in the range of about 4.5 to about 6, or about 5 to about 6, or about 5 to about 5.5. .. In some embodiments, the pharmaceutical product has a pH of 5.2.

0019In addition, chondropathy, clavicle skull dysplasia, endochondroma, fibrous bone dysplasia, Gauche's disease, hypophosphatemic ulcer, Malfan's syndrome, hereditary multiple external osteoma, neurofibrooma Disease, osteodysplasia, marble bone disease, osteomaculosis, sclerosing lesions, pseudojoints, purulent myelitis, periodontal disease, antiepilepopenia-induced bone loss, primary or secondary hyperthyroidism , Familial hyperthyroidism syndrome, gravity-induced bone loss, male osteopenia, postmenopausal bone loss, osteoarthritis, renal bone dystrophy, bone invasive disease, oral bone loss, jaw bone necrosis, Juvenile Paget's disease, meloleostosis, metabolic bone disease, obesity cytosis, sickle redemia / disease, organ transplant-related bone loss, kidney transplant-related bone loss, systemic erythematosus, tonic spondylitis, epilepsy, Juvenile arthritis, salacemia, mucopolysaccharidosis, Fabry's disease, Turner's syndrome, Down's syndrome, Kleinfelder's syndrome, Hansen's disease, Pertes' disease, adolescent idiopathic spinal kyphosis, infancy-onset multisystem inflammatory disease, Winchester's syndrome, Menquez's disease , Wilson's disease, ischemic bone disease (leg carpepertes disease or focal migratory osteoporosis, etc.), anemia, steroid-induced conditions, glucocorticoid-induced bone loss, heparin-induced bone loss, myelopathy, disruption Blood disease, malnutrition, calcium deficiency, osteoporosis, osteopenia, alcohol dependence, chronic liver disease, postmenopausal condition, chronic inflammatory condition, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, inflammatory colitis, Crohn's disease, rare menstruation, amenorrhea, pregnancy, diabetes, hyperthyroidism, thyroid disorders, parathyroid disorders, Cushing's disease, terminal hypertrophy, osteopenia, lack of exercise or inactivity, reflex sympathetic dystrophy Syndrome, focal osteoporosis, osteosoftness, osteopenia associated with joint replacement, osteopenia associated with HIV, osteopenia associated with growth hormone loss, osteopenia associated with cystic fibrosis, Chemotherapy-related bone loss, tumor-induced bone loss, cancer-related bone loss, hormone-removed bone loss, multiple myeloma, drug-induced bone loss, neurological ingestionDesire, disease-related facial bone loss, disease-related head bone loss, disease-related jaw bone loss, disease-related skull bone loss, aging-related bone loss, aging-related facial bone loss, aging-related Any disease associated with, but not limited to, head bone loss, aging-related jaw bone loss, aging-related skull loss, or space travel-related bone loss. Also described herein are methods of using the formulations described herein to be treated.

0020The formulations described herein, in some embodiments, include orthopedic surgery, dental surgery, implant surgery, joint replacement, bone grafting, bone cosmetic surgery, and fracture healing, non-adhesive healing, protracted healing, and. It is useful for improving the results of bone repair such as facial reconstruction. One or more formulations may be administered before, during, and / or after treatment, replacement, transplantation, surgery, or repair.

0021Such a method may include administering a therapeutically effective amount, for example, an amount of the preparation effective for improving bone density, and may further include administering a second therapeutic agent.

0022Also, to achieve a dose of about 0.5-20 mg, or 0.5-10 mg per kg body weight of a patient, with vials, kits, or containers containing the formulations described herein, eg, filled syringes or infusion devices, and optionally. A label containing instructions for using the appropriate volume or amount of the formulation required for the above is disclosed herein.

0023While the various embodiments herein are shown using the term "contains", under various circumstances, the relevant embodiments also include the term "consisting of" or "consisting of essentially". Please understand that it can be described using. It should be noted that the term "a" or "an" refers to one or more, for example, "an immunoglobulin molecule" is one or more immunoglobulin molecules. Is understood to represent. Therefore, the terms "a (or" an ")", "one or more", and "at least one" may be used interchangeably herein.

0024It should also be understood that when representing a range of values, the numbers represented can be individual values found within that range. For example, "pH of about pH 4 to about pH 6" can be, but is not limited to, pH 4, 4.2, 4.6, 5.1, 5.5, etc., and any value between such values. Furthermore, "pH of about pH 4 to about pH 6" should not be construed to mean that the pH of the product changes the unit of 2 pH in the range of pH 4 to pH 6 during storage, but rather one value. However, it may be selected in that range with respect to the pH of the solution, which remains approximately buffered at that pH. In some embodiments, when the term "about" is used, it means the listed number + or-5%, 10%, 15%, or more of the listed number. The actual variation of interest can be determined from the context.

0025In any of the ranges described herein, the endpoints of that range are included within that range. However, the representation also considers the same range excluding low and / or high endpoints. Further features and variations of the invention will be apparent to those skilled in the art from the entire application, including drawings and detailed description, and all such features are intended as aspects of the invention. Similarly, the features of the invention described herein are further intended as aspects of the invention as well, regardless of whether the combination of features is specifically described above as an aspect or embodiment of the invention. It can be rearranged into an embodiment. Also, only those limitations as described herein as essential to the present invention should be considered as such and do not include limitations not described herein as essential. Variants of the invention are intended as aspects of the invention.

0026The present article contains formulations containing high concentrations of antibodies containing calcium salts and / or acetates or buffers to reduce viscosity, methods of using these formulations, and containers or kits containing these formulations. Describe in the specification.

0027<u style="single"> I. Antibodies in the product</u> Present at concentrations of g / mL, about 155 mg / mL, about 156 mg / mL, about 157 mg / mL, about 158 mg / mL, about 159 mg / mL, or about 160 mg / mL, up to, for example, about 300 mg / mL, About 290 mg / mL, about 280 mg / mL, about 270 mg / mL, about 260 mg / mL, about 250 mg / mL, about 240 mg / mL, about 230 mg / mL, about 220 mg / mL, about 210 mg / mL, about 200 mg / mL, It may range to about 190 mg / mL, about 180 mg / mL, or even about 170 mg / mL. Approximately 70 mg / mL to approximately 250 mg / mL, approximately 70 mg / mL to approximately 200 mg / mL, approximately 70 mg / mL to approximately 160 mg / mL, approximately 100 mg / mL to approximately 250 mg / mL, approximately 100 mg / mL to approximately 200 mg / mL, Alternatively, any range characterizing the combination of the above endpoints is considered, including but not limited to about 100 mg / mL to about 180 mg / mL.

0028Antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab -9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab-21 , Ab-22, Ab-23, and Ab-24 have already been described in U.S. Patent Application Publication No. 2007/0110747, the disclosure of which, including the sequence listing, is incorporated herein by reference in its entirety. ..

0029The anti-sclerostin antibodies described herein are 10^-6Or less, or 10^-7Or less, or 10^-8Or less, or 10^-9Or less K_DAnd binds to sclerostin of SEQ ID NO: 1 (lower numbers mean higher binding affinity). Affinity can be determined by any means known in the art, including those by Biacore technology.

0030In some exemplary embodiments, the antibody is the antibody Ab-A, Ab-B, Ab-C, Ab-D, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6. , Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-15, Ab-16, Ab-19, Ab-23, or Ab-24 Includes heavy and / or light chains. Antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, including constant regions The amino acid sequences of the mature full-length light chains of Ab-9, Ab-10, Ab-11, Ab-12, Ab-15, Ab-16, Ab-17, Ab-19, Ab-23, and Ab-24 are: , SEQ ID NOs: 8, 22, 32, 42, 52, 62, 80, 88, 98, 108, 118, 128, 138, 148, 166, 176, 184, 70, 210 It is described in 222 and 246, respectively. Antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6 , Ab-7, Ab-8 , including constant regions , Ab-9, Ab-10, Ab-11, Ab-12, Ab-15, Ab-16, Ab-19, Ab-23, and Ab-24. 10, 24, 34, 44, 54, 64, 82, 90, 100, 110, 120, 130, 140, 150, 168, 178, 186, 72, 224, and 248, respectively.

0031Antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, including constant regions The corresponding cDNA sequences encoding the full-length light chains of Ab-9, Ab-10, Ab-11, Ab-12, Ab-15, Ab-16, Ab-19, Ab-23, and Ab-24 , SEQ ID NOs: 7, 21, 31, 41, 51, 61, 79, 87, 97, 107, 117, 127, 137, 147, 165, 175, 183, 69, 209, 221, and 245, respectively. .. Antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab The corresponding cDNA sequence, which encodes a full-length heavy chain containing the constant regions of -10, Ab-11, Ab-12, Ab-15, Ab-16, Ab-19, Ab-23, and Ab-24, is It is set forth in SEQ ID NOs: 9, 23, 33, 43, 53, 63, 81, 89, 99, 109, 119, 129, 139, 149, 167, 177, 185, 71, 211, 223, and 247, respectively.

0032In other exemplary embodiments, the antibody comprises antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-15, Ab-16, Ab-17, Ab-19, Ab-21, Ab-23, or Ab Includes heavy and / or light chain variable regions of any of -24. For example, the antibody has SEQ ID NO: 14 (Ab-1 heavy chain variable region) and / or SEQ ID NO: 12 (Ab-1 light chain variable region), SEQ ID NO: 68 (Ab-15 heavy chain variable region) and / or sequence. No. 66 (Ab-15 light chain variable region), or SEQ ID NO: 86 (Ab-5 heavy chain variable region) and / or SEQ ID NO: 84 (Ab-5 light chain variable region), or SEQ ID NO: 154 (Ab-16 heavy) Chain variable region) and / or SEQ ID NO: 152 (Ab-16 light chain variable region), or SEQ ID NO: 182 (Ab-14 heavy chain variable region) and / or SEQ ID NO: 180 (Ab-14 light chain variable region), or SEQ ID NO: 208 (Ab-19 heavy chain variable region) and / or SEQ ID NO: 207 (Ab-19 light chain variable region), or SEQ ID NO: 216 (Ab-20 heavy chain variable region) and / or SEQ ID NO: 214 (Ab- 20 light chain variable region), or SEQ ID NO: 220 (Ab-23 heavy chain variable region) and / or SEQ ID NO: 218 (Ab-23 light chain variable region), or SEQ ID NO: 238 (Ab-22 heavy chain variable region) and / Or contains SEQ ID NO: 236 (Ab-22 light chain variable region).

0033In some embodiments, the antibody has SEQ ID NOs: 1-5 (Ab-A and Ab-1 CDRs), or 15-20 (Ab-B CDRs), or 25-30 (Ab-C CDRs), or 35-40 (Ab-D CDR), or 45-50 (Ab-2 CDR), or 55-60 (Ab-3 and Ab-15 CDR), or 73-78 (Ab-4 and Ab-5 CDR) , 91-96 (Ab-6 CDR), or 101-106 (Ab-7 CDR), or 111-116 (Ab-8 CDR), or 121-126 (Ab-9 CDR), or 131-136 ( Ab-10 CDR), or 141-146 (Ab-11 and Ab-16 CDR), or 159-164 (Ab-12 CDR), or 169-174 (Ab-13 and Ab-14 CDR), or 187 ~ 192 (Ab-17 and Ab-18 CDRs), or 201-206 (Ab-19, Ab-20, and Ab-23 CDRs), or 225-229 (Ab-21 and Ab-22 CDRs), or 239 ~ 244 (Ab-24) Contains the CDRs described in CDR).

0034In some embodiments, the antibody comprises the antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-2, Ab-3, Ab-4, Ab-5 described herein. , Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-15, Ab-16, Ab-19, Ab-23, or Ab-24 It comprises an amino acid sequence obtained by expressing a cDNA encoding any of the heavy and / or light chains, or heavy and / or light chain variable regions, in a mammalian host cell. In any of the formulations described herein, in some embodiments, the antibody is a tetrameric immunoglobulin consisting of two heavy chains and two light chains.

0035In some embodiments, the antibody comprises the antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab- 7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-15, Ab-16, Ab-19, Ab-23, or Ab-24 CDR Including, antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab, respectively. At least 75%, 80% with heavy and / or light chains of -9, Ab-10, Ab-11, Ab-12, Ab-15, Ab-16, Ab-19, Ab-23, or Ab-24. , 85%, 90%, 95%, 96%, 97%, 98%, or 99% Includes heavy and / or light chains containing identical amino acid sequences. In some embodiments, the antibody comprises the antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab- 7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-15, Ab-16, Ab-19, Ab-23, or Ab-24 CDR Including, antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab, respectively. At least 75% of the heavy and / or light chain variable regions of -9, Ab-10, Ab-11, Ab-12, Ab-15, Ab-16, Ab-19, Ab-23, or Ab-24, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% Includes heavy and / or light chains containing identical amino acid sequences.

0036In some embodiments, the antibody is:

00371) Antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8 , Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab -21, Ab-22, Ab-23, or Ab-24, any one, two, three, four of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and / or CDRL3 It retains one, five, or six and optionally contains one or two mutations in such a CDR.

00382) Antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8 , Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab Holds all of the CDRH1, CDRH2, CDRH3, or heavy chain variable regions of any of -21, Ab-22, Ab-23, or Ab-24, and optionally one or one in such a CDR. Includes two mutations.

00393) Antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8 , Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab-18, Ab-19, Ab-20, Ab Holds all of the CDRL1, CDRL2, CDRL3, or light chain variable regions of any of -21, Ab-22, Ab-23, or Ab-24, and optionally one or one in such a CDR. Includes two mutations.

00404) For example, antibodies determined by X-ray crystallography Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab-17, Ab- Of the same sclerostin as the amino acids in the loop formed by 18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, or Ab-24, or amino acids 86-111 of SEQ ID NO: 249. It binds to an epitope. And / or

00415) Approximately 75%, over 80%, or approximately 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, or more than 95%, to bind to sclerostin, antibodies Ab-A, Ab-B, Ab-C, Ab-D, Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, Ab-9, Ab-10, Ab-11, Ab-12, Ab-13, Ab-14, Ab-15, Ab-16, Ab- Compete with 17, Ab-18, Ab-19, Ab-20, Ab-21, Ab-22, Ab-23, or Ab-24.

0042In some embodiments, the antibody comprises all three light chain CDRs, mature light chain variable regions, all three heavy chain CDRs, mature heavy chain variable regions, all six CDRs, or mature light chains and Includes both mature heavy chain variable regions. In some exemplary embodiments, two light chain CDRs from one antibody may be combined with a third light chain CDR from different antibodies. Alternatively, CDRL1 from one antibody can be combined with CDRL2 from different antibodies, and CDRL3 from yet another antibody, especially if the CDRs show a high degree of homology. Similarly, two heavy chain CDRs from one antibody may be combined with a third heavy chain CDR from different antibodies, or CDRH1 from one antibody, especially CDRs, have a high degree of homology. When indicated, it can be combined with CDRH2 from different antibodies, and CDRH3 from yet another antibody.

0043The term "antibody" refers to an intact antibody or binding fragment thereof. Antibodies may include complete antibody molecules, including polyclonal, monoclonal, chimeric, humanized, or human forms having full long and / or light chains, or antigen-binding fragments thereof. The antibody fragment is F (ab')₂, Fab, Fab', Fv, Fc, and Fd fragments, including single-domain antibody, single-chain antibody, maxibody, minibody, intrabody, diabody, tribody, tetrabody, v-NAR, and bis. -Can be incorporated into scFv (see, eg, Hollinger and Hudson, Nature Biotechnology, 23 (9): 1126-1136 (2005)).

0044An "isolated" antibody is an antibody that has been identified and isolated from its natural environment components, as the term is defined herein. Contaminating components of the natural environment are substances that interfere with the diagnostic or therapeutic use of antibodies and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In certain embodiments, the antibody is (1) to obtain at least 15 residues of the N-terminal or internal amino acid sequence, up to&gt; 95% by weight, and most preferably&gt; 99% by weight, of the antibody. Purification to a sufficient degree, or (3) Coomassie blue, or preferably by SDS-PAGE under reducing or non-reducing conditions using silver staining. An isolated native antibody contains an in situ antibody within a recombinant cell because at least one component of the antibody's natural environment is absent. However, isolated antibodies are usually prepared by at least one purification step.

0045An "immunoglobulin" or "natural antibody" is a tetrameric glycoprotein. In natural immunoglobulins, each tetramer consists of two homologous pairs of polypeptide chains, each pair being one "light chain" (about 25 kDa) and one "heavy chain" (about 50-70 kDa). Has. The amino-terminal portion of each chain contains the "variable" ("V") region of about 100-110 or more amino acids that are primarily involved in antigen recognition. The carboxy-terminal portion of each chain defines a constant region that is primarily involved in effector function. Immunoglobulins can be divided into different classes depending on the amino acid sequence of their heavy chain constant domains. Heavy chains are classified into mu (μ), delta (Δ), gamma (γ), alpha (α), and epsilon (ε), respectively, as antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. Is defined. Some of these can be further subdivided into subclasses or isotypes such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. Different isotypes have different effector functions. For example, IgG1 and IgG3 isotypes have antibody-dependent cellular cytotoxicity (ADCC) activity. Human light chains are classified into kappa (κ) and lambda (λ) light chains. Within the light and heavy chains, the variable and constant regions are linked by the "J" region of about 12 or more amino acids, and the heavy chain is also the "D" region of about 10 or more amino acids. Including. In general, see Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)).

0046Allotypes may be immunogenic and are often changes in antibody sequences in constant regions encoded by specific alleles in humans. Allotypes have been identified for five of the human IGHC genes, the IGHG1, IGHG2, IGHG3, IGHA2, and IGHE genes, represented as G1m, G2m, G3m, A2m, and Em allotypes, respectively. At least 18 Gm allotypes are known: nG1m (1), nG1m (2), G1m (1, 2, 3, 17) or G1m (a, x, f, z), G2m (23) or G2m (n) ), G3m (5, 6, 10, 11, 13, 14, 15, 16, 21, 24, 26, 27, 28) or G3m (b1, c3, b5, b0, b3, b4, s, t, g1 , C5, u, v, g5). There are two A2m allotypes, A2m (1) and A2m (2).

0047The term "hypervariable" region refers to amino acid residues from complementarity determining regions or CDRs (ie, Kabat et al., Sequences of Proteins of Immunological Interest, 5).^th Residues 24-34 (L1), 50-56 (L2), and 89- in the light chain variable domain, as described by Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991). 97 (L3), as well as 31-35 (H1), 50-65 (H2), and 95-102 (H3)) in heavy chain variable domains. Even a single CDR can recognize and bind antigens, albeit with a lower affinity than the full antigen binding site that contains all of the CDRs.

0048Alternative definitions of residues from the hypervariable "loop" are 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain, and in the heavy chain variable domain. They are described by Chothia et al., J. Mol. Biol. 196: 901-917 (1987) as 26-32 (H1), 53-55 (H2), and 96-101 (H3).

0049A "skeleton" or FR residue is a variable region residue other than a hypervariable region residue.

0050An "antibody fragment" comprises a portion of an intact immunoglobulin, preferably an antigen binding or variable region of an intact antibody, and comprises a multispecific (bispecific, trispecific, etc.) antibody formed from the antibody fragment. .. The immunoglobulin fragment may be produced by recombinant DNA technology or by enzymatic or chemical cleavage of the intact antibody.

0051Examples of unlimited examples of antibody fragments include Fab, Fab', F (ab').₂, Fv (variable region), domain antibody (containing dAb, VH domain) (Ward et al., Nature 341: 544-546, 1989), complementarity determining region (CDR) fragment, single chain antibody (scFv,) Contains VH and VL domains on a single polypeptide chain) (Bird et al., Science 242: 423-426, 1988, and Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879- 5883,1988, optionally containing a polypeptide linker, and optionally, multispecific, Gruber et al., J. Immunol. 152: 5368 (1994)), single chain antibody fragment, diabody (of another strand). VH and VL domains on a single polypeptide chain paired with complementary VL and VH domains) (European Patent EP404,097, WO93 / 11161, and Hollinger et. al., Proc.Natl.Acad.Sci.USA, 90: 6444-6448 (1993)), triabodies, tetrabodies, minibodies (via peptide linker (no hinge) or via IgG hinges to CH3 Fused scFv) (Olafsen, et al., Protein Eng Des Sel. 2004 Apr; 17 (4): 315-23), linear antibody (tandem Fd fragment (VH-CH1-VH-CH1)) (Zapata et al) ., Protein Eng., 8 (10): 1057-1062 (1995)), Chelate recombinant antibody (crAb, capable of binding to two adjacent epitopes on the same antigen) (Neri et al., J Mol Biol. 246) : 367-73,1995), bibody (bispecific Fab-scFv) or tribody (trispecific Fab- (scFv) (2)) (Schoonjans et al., J Immunol. 165: 7050-57,2000, Willems et al., J Chromatogr B Analyt Technol Biomed Life Sci.786: 161-76,2003), Intrabody (Biocca, et al., EMBO J.9: 101-108,1990, Colby et al., Proc Natl Acad Sci US A.101: 17616-21, 2004) (Can also include cellular signal sequences that carry or induce antibodies intracellularly (Mhashilkar et al, EMBO J 14: 1542-51, 1995, Wheeler et al., FASEB J. 17: 1733-5, 2003). ), Transbodies (cell-permeable antibodies containing protein transfection domains (PTDs) that fuse with scFv) (Heng et al., Med Hypotheses. 64: 1105-8,2005), Nanobodies (about 15 kDa heavy chain variables) Domain) (Cortez-Retamozo et al., Cancer Research 64: 2853-57,2004), Small Module Immunopharmaceutical (SMIP) (International Publication No. WO 03/041600, US Patent Publication No. 200303133939, and US Patent Publication No. 200301118592), Antigen Binding Domain Immunoglobulin Fusion Protein, Camel Antibodies (where VH recombines with constant regions containing hinges, CH1, CH2, and CH3 domains) (Desmyter et al., J. Biol. Chem. 276: 26285-90, 2001, Ewert et. al., Biochemistry 41: 3628-36,2002, US Patent Publication Nos. 20050136049 and 20050037421), VHH-containing antibodies, heavy chain antibodies (HCAb, two heavy chain homodimers with structure H2L2), and also. A polypeptide containing at least a portion of an immunoglobulin that is sufficient to provide a specific antigen that binds to a polypeptide, such as a CDR sequence, as long as the variant or derivative, as well as the antibody, retains the desired biological activity. Can be mentioned.

0052The term "mutant" used in connection with an antibody refers to at least one amino acid in the variable region and the portion corresponding to the variable region, provided that the mutant retains the desired binding affinity or biological activity. Refers to the polypeptide sequence of an antibody that contains a substitution, deletion, or insertion. In addition, the antibodies described herein may have amino acid modifications in the constant region to modify the effector function of the antibody, including half-life or clearance, ADCC and / or CDC activity. Such modifications can enhance pharmacokinetics or enhance antibody efficacy, for example, in treating cancer. Shields et See al., J. Biol. Chem., 276 (9): 6591-6604 (2001), which is incorporated herein by reference in its entirety. For IgG1, modifications to the constant region, especially the hinge or CH2 region, can increase or decrease effector function, including ADCC and / or CDC activity. In other embodiments, the IgG2 constant region is modified to reduce antibody-antigen aggregate formation. In the case of IgG4, modifications to the constant region, especially the hinge region, can reduce the formation of semi-antibodies.

0053The term "modification" used in connection with the antibodies or polypeptides described herein refers to one or more amino acid changes (including substitutions, insertions, or deletions), chemical modifications that do not interfere with hepsidin-binding activity. , Covalent modification by conjugation to therapeutic or diagnostic agents, labeling (eg, by radioactive nuclei or various enzymes), binding of covalent polymers such as pegation (derivative with polyethylene glycol), and non-natural Includes, but is not limited to, chemical synthetic insertions or substitutions of amino acids. In some embodiments, the modified polypeptides of the invention, including antibodies, will retain the binding properties of the unmodified molecules of the invention.

0054The term "derivative" used in connection with the antibodies or polypeptides of the invention refers to covalent modification, labeling (eg, by radioactive nuclei or various enzymes) by conjugation to a therapeutic or diagnostic agent. Refers to an antibody or polypeptide that is covalently modified by binding a covalent polymer such as pegation (derivative with polyethylene glycol) and by inserting or substituting an unnatural amino acid by chemical synthesis. In some embodiments, the derivatives of the invention will retain the binding properties of the non-derivatized molecules of the invention.

0055Methods for making bispecific or other multispecific antibodies are known in the art and are chemically crosslinked, using leucine zippers (Kostelny et al., J. Immunol. 148: 1547-). 1553,1992), Diabody technology (Hollinger et al., Proc.Natl.Acad.Sci.USA 90: 6444-48,1993), scFv dimer (Gruber et al., J.Immunol.152: 5368, 1994), linear antibodies (Zapata et al., Protein Eng. 8: 1057-62, 1995), and chelated recombinant antibodies (Neri et al., J Mol Biol. 246: 367-73, 1995). ..

0056Protein and non-protein agents may be conjugated to antibodies by methods known in the art. Conjugation methods include direct binding, binding via a covalent linker, and specific binding pair members (eg, avidin-biotin). Such methods include, for example, those described by Greenfield et al., Cancer Research 50,6600-6607 (1990) for the conjugate of doxorubicin, and Arnon et al., Adv.Exp for the conjugate of platinum compounds. Examples include those described by .Med.Biol.303,79-90 (1991) and Kiseleva et al., Mol.Biol. (USSR) 25,508-514 (1991).

0057In some embodiments, the antibodies and antibody fragments described herein are obtained, for example, from natural antibodies, or Fab or scFv phage display libraries. The phrase "humanized antibody" refers to an antibody derived from a non-human antibody, typically a rodent monoclonal antibody, which includes modifications that bring the sequence to a more human-like state. Alternatively, the humanized antibody may be derived from a chimeric antibody.

0058The antibody fragment is V_HA pair of tandem Fd fragments (V) containing a domain antibody (dAb) fragment consisting of a domain (Ward et al., Nature 341: 544-546, 1989), the "linear antibody" forming a pair of antigen binding regions._H-C_H1-V_H-C_H1) is included. Linear antibodies can be bispecific or monospecific (Zepata et al. Protein Eng. 8: 1057-62 (1995)). A "minibody" consisting of scFv fused to CH3 via a peptide linker (without hinges) or via an IgG hinge is a "minibody" consisting of Olafsen, et al., Protein Eng Des Sel. 2004 Apr; 17 (4): It is described in 315-23. "Maxibody" refers to a divalent scFv covalently bound to the Fc region of an immunoglobulin, eg Fredericks et al, Protein Engineering, Design &amp; Selection, 17: 95-106 (2004) and Powers et al. , Journal of Immunological Methods, 251: 123-135 (2001). Heavy chain antibodies such as VH_HDomain, or H₂L₂(Called "heavy chain antibody" or "HCAbs"), or camelized V_HH(For example, Reichman, et al., J Immunol Methods 1999, 231: 25-38, Desmyter et al., J. Biol. Chem. 276: 26285-90, 2001, Ewert et al., Biochemistry 41: 3628-36, See 2002), Nanobodies (Cortez-Retamozo et al., Cancer Research 64: 2853-57, 2004). Intrabody is a single-chain antibody that exhibits intracellular expression and is capable of manipulating intracellular protein function (Biocca, et al., EMBO J. 9: 101-108,1990, Colby et al., Proc Natl Acad Sci USA. 101: 17616-21,2004, Mahashilkar et al, EMBO J 14: 1542-51, 1995, Wheeler et al. (FASEB) J.17: 1733-5.2003)). Transbody is a cell-permeable antibody in which the protein transduction domain (PTD) is fused with a single-chain variable fragment (scFv) antibody (Heng et al., (Med Hypotheses. 64: 1105-8, 2005). )). A binding domain immunoglobulin fusion protein specific for SMIP, or target protein, is a single-stranded polypeptide comprising an antigen-binding domain that fuses with the immunoglobulin domain required to perform antibody effector function. See, for example, International Publication No. WO 03/041600, US Patent Publication No. 20030133939, and US Patent Publication No. 200301118592.

0059<u style="single">II. Calcium and acetate or buffer</u> It has been found that adding a relatively low concentration of calcium acetate to the formulation of the selected antibody reduces the viscosity of the formulation. The term "viscosity" used herein refers to "absolute viscosity". Absolute viscosity, sometimes referred to as dynamic or simple viscosity, is the product of kinematic viscosity and fluid density: absolute viscosity = kinematic viscosity x density. The dimension of kinematic viscosity is L²/ T, L is the length, and T is the time. Generally, the kinematic viscosity is expressed in centistoke (cSt). The SI unit of kinematic viscosity is mm²/ s, which is 1cSt. Absolute viscosity is expressed in units of centipores (cP). The SI unit of absolute viscosity is millipascal-seconds (mPa-s) and 1cP = 1mPa-s.

0060Such viscosity measurements are made hourly (eg, 1-23 hours), daily (eg, 1-10 days), weekly (eg, 1-5 weeks) after the addition of the viscosity reducer to the antibody formulation. , Or monthly (eg, 1-12 months), or yearly (eg, 1-2 years, 1-3 years). Viscosity measurements may be made at storage or dosing temperature, eg 2-8 ° C or 25 ° C (room temperature). In some embodiments, the absolute viscosity of the liquid or reconstituted liquid formulation at storage and / or administration temperature is 15 cP or less, or 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, Or it is 4cP or less.

0061In some embodiments, the viscosity of the protein formulation is measured before and after the addition of calcium and / or acetate (and / or buffer). Methods of measuring viscosity are well known in the art and include, for example, the use of capillary viscometers or cone plate rheometers. Any method may be used provided that the same method is used to compare the test and control formulations.

0062The viscosity of the antibody preparation can be reduced by adding a calcium salt and / or acetate (and / or buffer) to the preparation. The viscosity of the antibody preparation is about 5%, about 10%, about 15%, about 20% compared to the viscosity of the comparative antibody preparation, which does not contain calcium salt and / or acetate (and / or buffer). , About 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about It can be reduced by 85%, and about 90%.

0063Exemplary calcium salts include, but are not limited to, calcium acetate, calcium carbonate, and calcium chloride. In some embodiments, the calcium salt has a concentration of at least 0.5 mM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM. In certain embodiments, the calcium salt concentration is 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, or 25 mM or less. Any range characterizing the combination of the above endpoints is considered, including but not limited to about 0.5 mM to about 10 mM, about 5 mM to about 10 mM, or about 5 mM to about 15 mM. In some embodiments, the calcium salt is at least 30%, 40%, 50%, 60%, or more of the antibody formulation as compared to the same formulation of the antibody that does not contain acetate and / or buffer. It is present at a concentration that reduces the viscosity or achieves a viscosity of 10 cP or less, or 9, 8, 7, 6, or 5 cP or less. In certain embodiments, the calcium salt is added at a low concentration so that it does not adversely affect the protein formulation. For example, at calcium or magnesium chloride concentrations of 20 mM or higher, proteins can form gels at low storage temperatures (2-8 ° C). Therefore, the concentration of calcium salt reduced at the desired storage temperature of the reduced viscosity formulation is generally selected.

0064In all of the ranges described herein, the concentration of a cation, anion, or salt described is the final concentration in the liquid or reconstituted liquid formulation to be administered. In any of the ranges described herein, the endpoints of the range are included within that range. However, the representation also considers the same range excluding low and / or high endpoints.

0065In some embodiments, the formulations described herein further comprise, in addition to the calcium salt, an acetate buffer at a concentration of at least 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, or 15 mM. In some embodiments, the concentration is 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM or less. Any range characterizing the combination of the above endpoints is considered, including but not limited to about 5 mM to about 15 mM, or about 5 mM to about 10 mM. The buffer is preferably added at a concentration that maintains the pH at about 5-6, or 5-5.5, or 4.5-5.5. When the calcium salt in the formulation is calcium acetate, in some embodiments the total concentration of acetate is from about 10 mM to about 50 mM, or from about 20 mM to about 40 mM.

0066In some embodiments, the pharmaceutical product has a total concentration of acetate, which is at least about 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, or 50 mM. In some embodiments, the acetate concentration is about 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, or 90 mM or less. Consider any range that characterizes the combination of the above endpoints, including but not limited to about 10 mM to about 50 mM, about 20 mM to about 50 mM, about 20 mM to about 40 mM, about 30 mM to about 50 mM, or about 30 mM to about 75 mM. Will be done. In some embodiments, the acetate or buffer comprises calcium acetate and / or sodium acetate. Alternatively, in some embodiments, the acetate and / or buffer is at least 30%, 40%, 50%, 60%, as compared to the same formulation of the acetate and / or buffer-free antibody. Or more, it is present at a concentration that reduces the viscosity of the antibody formulation or achieves a viscosity of 10 cP or less, or 9, 8, 7, 6, or 5 cP or less. As a non-limiting example, a solution containing 10 mM calcium acetate has 20 mM acetate anion and 10 mM calcium cation due to the divalent nature of the calcium cation, while 10 mM sodium acetate. The solution contained has 10 mM sodium cations and 10 mM acetate anions.

0067In some embodiments, the total concentration of ions (cations and anions) in solution is at least 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, It is 75 mM, 80 mM, or 85 mM. In some embodiments, the total concentration of ions is about 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM or less. Any combination of endpoints characterized by, but not limited to, about 30 mM to about 60 mM, or about 30 mM to about 70 mM, or about 30 mM to about 80 mM, or about 40 mM to about 150 mM, or about 50 mM to about 150 mM. The range is also taken into account. As a non-limiting example, a solution of 10 mM calcium acetate has a total concentration of 30 mM ions (10 mM cations and 20 mM anions).

0068In any of the formulations described herein, in some embodiments, the total osmolal concentration is 500 mOsm / L, 450 mOsm / L, 400 mOsm / L, or 350 mOsm / L or less, preferably, etc. It is close to Zhang, for example, 250-350m Osm / L.

0069In addition, other excipients known in the art or described herein can be included in the formulation.

0070<u style="single">III. Excipients in the formulation</u> Protein preparations are generally administered parenterally. If administered parenterally, they must be sterilized. Sterile diluents include liquids that are pharmaceutically acceptable (safe and non-toxic to human administration) and useful for the preparation of liquid formulations, such as formulation reconstitution after lyophilization. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffer (eg, phosphate buffered saline), sterile saline, Ringer solution, or dextrose solution. Diluents can include aqueous salts and / or buffers.

0071Excipients are additives that are included in the present formulation because they provide or enhance the stability, delivery, and manufacturability of the drug product. Excipients, regardless of why they are included, are an integral part of the drug product and therefore need to be safe and well tolerated by the patient. For protein drugs, the choice of excipient is particularly important as the excipient can affect both the efficacy and immunogenicity of the drug. Therefore, protein formulations need to be developed with the proper selection of excipients that provide suitable stability, safety, and marketability.

0072Excipients described herein are organized by their chemotype or their functional role in the formulation. When considering the type of each excipient, a brief description of the stabilization method is provided. Given the teachings and guidance provided herein, one of ordinary skill in the art would be able to readily change the amount and range of excipients without increasing the viscosity to undesired levels. Excipients are subject to the desired osmolality of the final solution (ie isotonic, hypotonic or hypertonic), pH, desired stability, resistance to aggregation or degradation or precipitation, freezing, lyophilization, or high temperature conditions. May be chosen to achieve protection in, or other properties. Various types of excipients are known in the art. Exemplary excipients include salts, amino acids, other tonicity agents, surfactants, stabilizers, fillers, antifreezes, rioprotectants, antioxidants, metal ions, chelating agents, and / Alternatively, preservatives may be mentioned.

0073Furthermore, if a particular excipient is reported in the formulation, eg, in percent (%) w / v, one of ordinary skill in the art will recognize that the equivalent molar concentration of that excipient is also considered. Will.

0074<u style="single">A. Buffer solution</u> Optimal stability pH ranges need to be identified early during prescription studies. Several methods, such as accelerated stability testing and calorimetric screening, have proven useful in this attempt (Remmele RLJr., Et al., Biochemistry, 38 (16): 5241-7 (1999). ). Once the formulation is finished, the drug product must be manufactured and maintained within the prescribed specifications throughout its shelf life. Therefore, in most cases buffers are employed to regulate the pH in the formulation.

0075Organic acids, phosphates, and tris have been commonly used as buffers in protein formulations (Table 1). The buffer capacity of buffer species is maximal at a pH equal to pKa and decreases as pH increases or decreases from this value. 90% of the buffer capacity is within 1 pH unit of its pKa. Buffer capacity also increases proportionally with increasing buffer concentration.

0076Several factors need to be considered when choosing a buffer. First and foremost, the buffer species and its concentration need to be defined based on their pKa and the desired pharmaceutical pH. It is equally important to ensure that the buffer is compatible with protein drugs, other pharmaceutical excipients and does not catalyze any degradation reactions. Recently, it has been shown that polyanionic carboxylic acid buffers such as citrate and succinate form covalent adducts with side chain residues of proteins. A third important feature to consider is the sensation of sting and irritation that the buffer can induce. For example, citrate is known to cause stinging at the time of injection (Laursen T, et al., Basic Clin Pharmacol). Toxicol., 98 (2): 218-21 (2006)). For drugs administered via the subcutaneous and intramuscular routes, where the drug solution remains in the site for a relatively longer period of time than when the drug is administered by the intravenous route, which is rapidly diluted in the blood at the time of administration. The potential for stinging and irritation is greater. For formulations administered by direct intravenous injection, the total amount of buffer (and any other formulation component) needs to be monitored. For example, potassium ions administered in the form of potassium phosphate buffer have been reported to induce cardiovascular effects in patients (Hollander-Rodriguez JC, et al., Am. Fam. Physician., 73 (2): 283-90 (2006)). Table 1: Commonly used buffers and their pKa values<tables num="1"><img id="000002" he="67" wi="159" file="JP6174176B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

0077The buffer system present in the present formulation is selected to be physiologically compatible and to maintain the desired pH.

0078The pH buffering compound may be present in any amount suitable for maintaining the pH of the pharmaceutical product at a given level. The pH buffer, eg acetate, may be present at a concentration of 0.1 mM to 1000 mM (1M). In one embodiment, the pH buffer is at least 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, or 900 mM. In another embodiment, the pH buffer concentrations are 1, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 , 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, or 90 mM to 100 mM. In yet another embodiment, the pH buffer concentration is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, or 40 mM. It is ~ 50 mM. In yet another embodiment, the pH buffer concentration is 10 mM.

0079Other exemplary pH buffers used herein to buffer the formulation include glycine, glutamate, succinate, phosphate, acetate, and aspartate. Is not limited to these. Amino acids such as histidine and glutamic acid can also be used as buffers.

0080<u style="single">B. Stabilizers and fillers</u> Stabilizers include antifreeze agents, rioprotectants, and a group of compounds that can serve as glass-forming agents. Antifreezes act to stabilize proteins during or in the frozen state at low temperatures. Rioprotectants stabilize proteins in lyophilized solid dosage forms by preserving the natural conformational properties of the proteins during the lyophilization dehydration phase. Glass state properties have been categorized as "rugged" or "fragile" depending on their relaxation properties as a function of temperature. It is important that the antifreeze, rioprotectant, and glass-forming agent remain in the same phase as the protein to provide stability. Sugars, polymers, and polyols fall into this category and can sometimes play all three roles.

0081Polyols include a group of excipients including sugars (eg, mannitol, sucrose, sorbitol), as well as other polyhydric alcohols (eg, glycerol and propylene glycol). The polymer polyethylene glycol (PEG) falls into this category. Polyols are commonly used as stabilizing excipients and / or isotonic agents in both liquid and lyophilized parenteral protein formulations. Polyols can protect proteins from both physical and chemical degradation pathways.

0082Illustrative C3-C6 polyols include propylene glycol, glycerin (glycerol), treose, triol, erythrose, erythritol, ribose, arabinose, arabitol, lixose, martitol, sorbitol, sorbose, glucose, mannitol, mannitol, rebroth Examples thereof include dextrose, martose, fructose, fructose, xylitol, inositol, galactose, xylose, fructose, sucrose, 1,2,6-hexanetriol and the like. Higher sugars include dextran, propylene glycol, or polyethylene glycol. Reducing sugars such as fructose, maltose, or galactose oxidize more easily than non-reducing sugars. Further examples of sugar alcohols are glucitol, maltitol, lactitol, or isomaltulose. Further exemplary lioprotectants include glycerin and gelatin, and sugars include melibiose, melesitose, raffinose, mannotriose, and stachyose. Examples of reducing sugars include glucose, maltose, lactose, maltulose, isomaltulose, and lactulose. Examples of non-reducing sugars include non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other linear polyhydric alcohols. Examples of monoglycosides include compounds obtained by reducing disaccharides such as lactose, maltose, lactulose, and maltulose.

0083In some embodiments, the formulation described herein also comprises a stabilizer (or combination of stabilizers) added to the formulation. The term "stabilizer" is an excipient that can prevent aggregation or other physical decomposition as well as chemical decomposition in aqueous and solid states (eg, autolysis, deamidation, oxidation, etc.). Means. Stabilizers conventionally used in pharmaceutical compositions include sucrose; trehalose; mannose; maltose; lactose; glucose; raffinose; cellobiose; gentiobiose; isomaltose; arabinose; glucosamine; fructose; mannose; sorbitol; glycine; arginine HCL; Polyhydroxy compounds, including dextran, starch, hydroxyethyl starch, cyclodextrin, N-methylpyrrolidene, cellulose, hyaluronic acid, etc .; sodium chloride (Carpenter et al., Develop. Biol. Standard) 74: 225, (1991)), but not limited to these. In one embodiment, the stabilizer is incorporated at a concentration of about 0% to about 40% w / v. In another embodiment, the stabilizer is at least 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 30, or 40% w / v. In another embodiment, the stabilizer is incorporated at a concentration of about 1, 2, 3, 4, 5, 6, 7, 8, 9% to about 10% w / v. In yet another embodiment, the stabilizer is incorporated at a concentration of about 2% to about 6% w / v. In yet another embodiment, the stabilizer is incorporated at a concentration of about 4% w / v. In yet another embodiment, the stabilizer is incorporated at a concentration of about 6% w / v.

0084If necessary, the present formulation also contains an appropriate amount of filler and osmolality adjusting agent suitable for forming a lyophilized "cake". The filler may be either crystalline (eg, mannitol, glycine) or amorphous (eg, polymers such as sucrose, dextran, polyvinylpyrrolidone, carboxymethyl cellulose). Other exemplary fillers include lactose, sorbitol, trehalose, or xylitol. In a further embodiment, the filler is incorporated at a concentration of about 0% to about 10% w / v. In another embodiment, the filler is at least 0.2, 0.5, 0.7, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0. , Or at a concentration of 9.5% w / v. In a further embodiment, the filler is at a concentration of about 1,1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5% to 5.0% w / v so as to produce a cake that is mechanically and pharmaceutically suitable. It is captured.

0085<u style="single">C. Surfactant</u> Protein molecules are more likely to interact with surfaces at the air-liquid, vial-liquid, and liquid-liquid (silicone oil) interfaces that make them susceptible to adsorption and denaturation. This degradation pathway is determined in inverse proportion to protein concentration and has been found to result in either the formation of soluble and insoluble protein aggregates or the loss of protein from solution through adsorption to the surface. In addition to adsorption on the container surface, physical agitation exacerbates surface-induced degradation, as can occur during product delivery and handling.

0086Surfactants are commonly used in protein formulations to prevent surface-induced degradation. Surfactants are amphipathic molecules that have the ability to compete with proteins for interfacial positions. The hydrophobic portion of the surfactant molecule occupies the interface position (eg, air / liquid), while the hydrophilic portion of the molecule remains oriented towards the bulk solvent. At sufficient concentrations (typically the critical micelle concentration of detergents), the surface layer of detergent molecules acts to prevent protein molecules from adsorption at the interface. Thereby, surface-induced decomposition is minimized. The most commonly used surfactants are fatty acid esters of sorbitan polyethoxylates, namely polysorbate 20 and polysorbate 80. (For example, Avonex®, Neupogen®, Neulasta®). The two differ only in the length of the aliphatic chain that gives the molecule hydrophobicity from C-12 and C-18, respectively. Therefore, polysorbate 80 is more surface active than polysorbate 20 and has a lower critical micelle concentration. The surfactant poloxamer 188 has also been used in several commercially available liquid products such as Gonal-F®, Norditropin®, and Ovidrel®.

0087Detergents can also affect the thermodynamic conformational stability of proteins. Here again, the effect of a given excipient is protein specific. For example, polysorbate has been shown to reduce the stability of some proteins and increase the stability of others. Detergent protein destabilization can be theoretically explained in terms of the hydrophobic tail of the detergent molecule, which may be involved in specific binding to the partially or totally denatured protein state. These types of interactions can cause a transition of conformational equilibrium to a more swollen protein state (ie, complement the binding polysorbate and increase the exposure of the hydrophobic portion of the protein molecule). Alternatively, if the native state of the protein exhibits several hydrophobic surfaces, a detergent that binds to the native state can stabilize its conformation.

0088Another feature of polysorbates is that they are inherently prone to oxidative degradation. Often, as raw materials, they contain sufficient amounts of peroxide to cause the oxidation of protein residue side chains, especially methionine. The potential for oxidative damage resulting from the addition of stabilizers emphasizes that the lowest effective concentration of excipient should be used in the formulation. For surfactants, the effective concentration for a given protein depends on the stabilization mechanism. When the surfactant stabilization mechanism is related to the prevention of surface denaturation, it is assumed that the effective concentration is approximately the critical micelle concentration of the detergent. Conversely, when the stabilization mechanism is associated with a specific protein-detergent interaction, the effective detergent concentration is related to the protein concentration and the stoichiometry of the interaction (Randolph TW, et al., Pharm Biotechnol). ., 13: 159-75 (2002)).

0089In addition, an appropriate amount of surfactant may be added to prevent surface-related aggregation phenomena during freezing and drying (Chang, B, J. Pharm. Sci. 85: 1325, (1996)). Exemplary surfactants include anions, cations, nonionics, zwitterions, and amphoteric surfactants, including surfactants derived from natural amino acids. Anionic surfactants include sodium lauryl sulfate, sodium dioctyl sulfosuccinate and sodium dioctyl sulfonate, chenodeoxycholic acid, sodium N-lauroyl sarcosin salt, lithium dodecyl sulfate, sodium 1-octane sulfonate, sodium silicate hydrate. , Sodium deoxycholic acid, and sodium glycodeoxycholic acid salt, but are not limited thereto. Cationic surfactants include, but are not limited to, benzalkonium chloride or benzethonium chloride, cetylpyridinium chloride monohydrate, and hexadecyltrimethylammonium bromide. Zwitterionic surfactants include, but are not limited to, CHAPS, CHAPSO, SB3-10, and SB3-12. Nonionic surfactants include, but are not limited to, digitonin, Triton X-100, Triton X-114, TWEEN-20, and TWEEN-80. In another embodiment, the surfactants include lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50, and 60, glycerol monostearate, polysorbate 40, 60, 65, And 80, soy lecithin and other phospholipids (such as DOPC, DMPG, DMPC, and DOPG), sucrose fatty acid esters, methyl cellulose and carboxymethyl cellulose.

0090The formulations described herein may further contain these surfactants individually or as a mixture in different proportions. In one embodiment, the surfactant is incorporated at a concentration of about 0% to about 5% w / v. In another embodiment, the surfactant is at least 0.001, 0.002, 0.005, 0.007, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, Incorporated at concentrations of 2.5, 3.0, 3.5, 4.0, or 4.5% w / v. In another embodiment, the surfactant is incorporated at a concentration of about 0.001% to about 0.5% w / v. In yet another embodiment, the surfactant is incorporated at a concentration of about 0.004, 0.005, 0.007, 0.01, 0.05, or 0.1% w / v to about 0.2% w / v. In yet another embodiment, the surfactant is incorporated at a concentration of about 0.01% to about 0.1% w / v.

0091In some embodiments, the decrease in viscosity is achieved with relatively little or no surfactant, eg, 0.1% or less total surfactant, or 0.05% or less, or 0.01% or less.

0092<u style="single">D. Amino acids</u> Amino acids have been used for multiple purposes in protein formulations as buffers, fillers, stabilizers, and antioxidants. Histidine and glutamic acid are employed to buffer the protein formulation in the pH range 5.5-6.5 and 4.0-5.5, respectively. The imidazole group of histidine has a pKa = 6.0 and the carboxyl group of the glutamic acid side chain has a pKa of 4.3, making them suitable for buffering in their individual pH range. Glutamic acid is found in several formulations (eg, Stemgen®). Histidine is commonly found in commercially available protein formulations (eg, Xolair®, Herceptin®, Recombinate®). It provides a good alternative to citrate, a buffer that is known to cause stinging at the time of injection. Interestingly, histidine has also been reported to have a stabilizing effect when used in high concentrations, both in liquid and lyophilized presentations (Chen B, et al., Pharm). Res., 20 (12): 1952-60 (2003)). Histidine (up to 60 mM) was also found to reduce the viscosity of high-concentration formulations of this antibody. However, in the same test, the authors observed increased aggregation and discoloration in the histidine-containing formulation during the freeze-thaw test of the antibody in a stainless steel container. The authors attributed this to the effect of iron ions eluted from the corrosion of steel vessels. Another caveat with histidine is that it undergoes photooxidization in the presence of metal ions (Tomita M, et al., Biochemistry, 8 (12): 5149-60 (1969)). The use of methionine as an antioxidant in the formulation appears promising. It has been shown to be effective against a number of oxidative stresses (Lam XM, et al., J Pharm Sci., 86 (11): 1250-5 (1997)).

0093The amino acids glycine, proline, serine, and alanine stabilize the protein. Glycine is also a filler commonly used in lyophilized formulations (eg, Neumega®, Genotropin®, Humatrope®). Arginine has been shown to be an effective agent in inhibiting aggregation and has been used in both liquid and lyophilized formulations (eg, Activase®, Avonex®, Enbrel (eg). Registered trademark) Liquid).

0094<u style="single">E. Antioxidants</u> Oxidation of protein residues results from a number of different causes. Besides the addition of specific antioxidants, prevention of oxidative protein damage is due to a number of factors throughout the manufacturing process and product storage, including atmospheric oxygen, temperature, exposure to light, and chemical contamination. With good management. The most commonly used pharmaceutical antioxidants are reducing agents, oxygen / free radical scavengers, or chelating agents. Antioxidants in therapeutic protein formulations must be water soluble and remain active throughout the shelf life of the product. Reducing agents and oxygen / free radical scavengers work by removing reactive oxygen species in solution. Chelating agents such as EDTA can be effective by binding trace metal contaminants that promote free radical formation. For example, EDTA was used in a liquid formulation of acidic fibroblast growth factor to inhibit the oxidation of cysteine residues catalyzed by metal ions. EDTA has been used in commercial products such as Kineret® and Ontak®.

0095However, the antioxidant itself can induce other covalent or physical changes to the protein. Many such cases have been reported in the literature. Reducing agents (such as glutathione) can cause disruption of intramolecular disulfide bonds, which can result in disulfide shuffling. In the presence of transition metal ions, ascorbic acid and EDTA have been shown to promote methionine oxidation in numerous proteins and peptides (Akers MJ, and Defelippis MR. Peptides and Proteins as Parenteral Solutions. In: Pharmaceutical). Formulation Development of Peptides and Proteins.Sven Frokjaer, Lars Hovgaard, editors.Pharmaceutical Science.Taylor and Francis, UK (1999)), Francsson JR, J.Pharm.Sci.86 (9): 4046-1050 (1997), Yin J, et al., Pharm Res., 21 (12): 2377-83 (2004)). Sodium thiosulfate has been reported to reduce the level of light and temperature-induced methionine oxidation in rhuMab HER2. However, the formation of thiosulfate-protein adducts has also been reported in this study (Lam XM, Yang JY, et al., J Pharm Sci. 86 (11): 1250-5 (1997)). The selection of the appropriate antioxidant is made according to the specific stress and sensitivity of the protein.

0096<u style="single">F. Metal ion</u> In general, transition metal ions are undesirable in protein formulations because they can catalyze physical and chemical degradation reactions in proteins. However, when a specific metal ion is a cofactor for a protein, it is contained in the preparation, and when forming a coordination complex, it is contained in a suspension preparation of the protein (for example, a zinc suspension of insulin). .. In recent years, the use of magnesium ions (10-120 mM) has been proposed to inhibit the isomerization of aspartic acid to isoaspartic acid (International Publication No. WO2004 / 039337).

0097Two examples of metal ions giving stable or increased activity in proteins are human deoxyribonuclease (rhDNase, Pulmozyme®) and factor VIII. For rhDNase, Ca⁺²Ions (up to 100 mM) increased the stability of the enzyme through specific binding sites (Chen B, et al., J Pharm Sci., 88 (4): 477-82 (1999)). In fact, the removal of calcium ions from the solution with EGTA caused increased deamidation and aggregation. However, this effect is Ca⁺²It was observed only when ions were used. Other divalent cations (Mg⁺², Mn⁺², And Zn⁺²) Was found to destabilize rhDNase. A similar effect was observed with factor VIII. Ca⁺²And Sr⁺²While the ions stabilized the protein, Mg⁺², Mn⁺²And Zn⁺², Cu⁺², And Fe⁺²Ions such as destabilized the enzyme (Fatouros, A., et al., Int. J. Pharm., 155, 121-131 (1997). In a separate test using factor VIII, Al⁺³A significant increase in aggregation rate was observed in the presence of ions (Derrick TS, et al., J. Pharm. Sci., 93 (10): 2549-57 (2004)). The authors found that other excipients, such as buffer salts, are often Al.⁺³It points out that it is contaminated with ions and explains the need to use excipients of appropriate quality in the formulated products.

0098<u style="single">G. Preservatives</u> Preservatives are required when developing multiple-use parenteral formulations with more than one extraction from the same container. Their main function is to inhibit microbial growth and ensure product sterility throughout the shelf life or period of use of the drug product. Commonly used preservatives include phenol, benzyl alcohol, metacresol, alkylparabens such as methylparaben or propylparaben, benzalkonium chloride, and benzethonium chloride. Other examples of compounds having antibacterial and antiseptic activity include octadecyldimethylbenzylammonium chloride and hexamethonium chloride. Other types of preservatives include aromatic alcohols such as butyl alcohol, phenol, benzyl alcohol, atecol, resorcinol, cyclohexanol, 3-pentanol. Although preservatives have a long history of use, the development of protein formulations containing preservatives can be difficult. Preservatives most often have a destabilizing effect (aggregation) on proteins, which is a major factor limiting the use of preservatives in multi-dose protein formulations (Roy S, et al., J Pharm Sci., 94 (2): 382-96 (2005)).

0099The presentation of a multi-use pen-type syringe includes a preservative formulation. For example, stored hGH formulations are currently available on the market. Norditropin® (Liquid, Novo Nordisk), Nutropin AQ® (Liquid, Genentech), and Genotropin (Freeze-Dried-Dual Chamber Cartridge, Pharmacia &amp; Upjohn) contain phenol, while Somatrope ( Registered trademark) (Eli Lilly) is formulated with m-cresol.

0100Several aspects need to be considered during the development of the conservative dosage form. The concentration of effective preservatives in drug products must be optimized. This requires testing given preservatives in dosage forms that have a concentration range that provides antibacterial efficacy without compromising protein stability. For example, using differential scanning calorimetry (DSC), three preservatives were successfully screened in the development of liquid formulations for the interleukin-1 receptor (type I). Preservatives were ranked based on their effect on stability at concentrations commonly used in commercial products (Remmele RL Jr., et al., Pharm Res., 15 (2): 200- 8 (1998)).

0101Some preservatives can cause injection site reactions, which is another factor that needs to be considered when choosing a preservative. Clinical trials focused on the evaluation of preservatives and buffers in Norditropin found lower pain perception in formulations containing phenol and benzyl alcohol compared to formulations containing m-cresol. (Kappelgaard AM, Horm Res.62 Suppl 3: 98-103 (2004)). Interestingly, among the commonly used preservatives, benzyl alcohol has an anesthetic effect (Minogue SC, and Sun DA., Anesth Analg., 100 (3): 683-6 (2005)).

0102<u style="single">IV. Kit</u> As a further aspect, kits are described herein comprising one or more formulations described herein, packaged in a manner that facilitates their use for administration to a subject. In one embodiment, such a kit is packaged in a container such as a closed bottle, tube, disposable or multi-use vial, prefilled syringe, or prefilled infusion device, the formulation described herein (eg,). , And optionally a label describing the use of the compound or composition in the practice of the method) is affixed to the container or included in the packaging. .. In one aspect, the compound or composition is packaged in unit dosage form. The kit may further include suitable devices for administering the composition according to a particular route of administration. Preferably, the kit comprises a label that describes the use of the antibodies described herein, or the formulations described herein.

0103<u style="single">V. Dosage</u> The dosing regimen involved in the methods for treating the conditions described herein includes various factors that modify the action of the drug, such as the patient's age, condition, weight, gender, and eating habits, any infection. Determined by the attending physician, taking into account the severity of the disease, duration of administration, and other clinical factors. In various embodiments, the daily formulation ranges from 0.1 to 50 mg of antibody preparation per kilogram of body weight (calculating the mass of protein alone, without chemical modification). In some embodiments, the dose is about 0.5 mg / kg to 20 mg / kg, or about 0.5 to 10 mg / kg.

0104The product is generally administered parenterally, eg, intravenously, subcutaneously, intramuscularly, via an aerosol (intrapulmonary or inhaled administration), or via a depot for long-term release. In some embodiments, the pharmaceutical product is administered intravenously by bolus first and then by continuous infusion to maintain therapeutic circulating levels of the drug product. In other embodiments, the product is administered as a single dose. One of ordinary skill in the art will readily optimize the effective dose and dosing regimen as determined by good medical practice and the clinical condition of the individual patient. Dosing frequency depends on the pharmacokinetic parameters of the drug and the route of administration. The optimal pharmaceutical formulation will be determined by one of ordinary skill in the art depending on the route of administration and the desired dose. For example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA) 18042) Please refer to pages1435-1712, the disclosure of which is incorporated herein by reference. Such formulations may affect the physical condition, stability, in vivo release rate, and in vivo clearance rate of the administered drug. Depending on the route of administration, a suitable dose may be calculated according to body weight, body surface area, or organ size. Further refinement of the calculations required to determine the appropriate dose for treatment with each of the above formulations, in particular, the dose information and assays disclosed herein, as well as the human clinical trials described above. It is routinely performed by one of ordinary skill in the art, taking into account the pharmacokinetic data found in. Appropriate doses can be confirmed by the use of established assays to determine blood concentration doses, along with appropriate dose-response data. The final dosing regimen includes various factors that modify the action of the drug, such as the specific activity of the drug, the severity of the patient's injury and responsiveness, the patient's age, condition, weight, gender, and diet, any infection. Determined by the attending physician, taking into account the severity of the disease, duration of administration, and other clinical factors. As the study is conducted, more information will be revealed regarding appropriate dosage levels and duration of treatment for various diseases and conditions.

0105<u style="single">VI. Therapeutic use of the drug</u> The formulations described herein are useful for treating or preventing bone-related disorders such as bone-related disorders associated with abnormal osteoblast or osteoclast activity. In some embodiments, chondropathy, clavicle skull dysplasia, endochondroma, fibrous osteoopenia, Gauche's disease, hypophosphatemic ulcer, Malfan's syndrome, hereditary polylateral bone. Tumor, neurofibromatosis, osteodysplasia, marble bone disease, osteomaculosis, sclerosing lesions, pseudojoint, purulent myelitis, periodontal disease, antiepileptic drug-induced bone loss, primary or secondary Hyperthyroidism, familial hyperthyroidism, gravity-induced bone loss, male osteoporosis, postmenopausal bone loss, osteoarthritis, renal bone dystrophy, bone invasive disease, oral bone mass Loss, jaw bone necrosis, juvenile Paget's disease, meloleostosis, metabolic bone disease, obesity cytosis, sickle erythrocyte anemia / disease, organ transplant-related bone loss, kidney transplant-related bone loss, systemic erythematosus, tonic Sponditis, epilepsy, juvenile arthritis, salacemia, mucopolysaccharidosis, Fabry's disease, Turner's syndrome, Down's syndrome, Kleinfelder's syndrome, Hansen's disease, Pertes' disease, adolescent idiopathic spinal kyphosis, infancy-onset multisystem inflammatory disease, Winchester syndrome, Menquez's disease, Wilson's disease, ischemic bone disease (such as leg carpepertes disease and focal migratory osteoporosis), anemia, steroid-induced conditions, glucocorticoid-induced bone loss, heparin-induced bone loss , Myelopathy, necrosis, malnutrition, calcium deficiency, osteoporosis, osteopenia, alcohol dependence, chronic liver disease, postmenopausal condition, chronic inflammatory condition, rheumatoid arthritis, inflammatory bowel disease, ulcerative colitis, Inflammatory colitis, Crohn's disease, rare menstruation, amenorrhea, pregnancy, diabetes, hyperthyroidism, thyroid disorders, parathyroid disorders, Cushing's disease, terminal hypertrophy, gonad dysfunction, lack of exercise or inactivity, reflexes Sexual Sympathetic Dystrophy Syndrome, Local Osteoporosis, Osteopenia, Osteopenia Associated with Joint Replacement, Osteopenia Associated with HIV, Osteopenia Associated with Growth Hormone Decrease, Osteopenia Associated with Cystic Fibrosis Osteopenia

0106In some embodiments, the formulations described herein include orthopedic surgery, dental surgery, implant surgery, joint replacement, bone grafting, bone cosmetic surgery, and fracture healing, non-adhesive healing, protracted healing, and. It is useful for improving the results of bone repair such as facial reconstruction. One or more compositions may be administered before, during, and / or after treatment, replacement, transplantation, surgery, or repair.

0107The product does not need to cure the subject of the disorder or completely protect it from the development of bone-related disorders in order to achieve a beneficial biological response. This product may be used prophylactically for the purpose of total or partial protection from bone-related disorders or their symptoms. This product is also used therapeutically to improve the bone-related disorder or its symptoms in whole or in part, or to protect the bone-related disorder or its symptoms from further progression in whole or in part. obtain. In fact, the materials and methods of the present invention are particularly useful for increasing bone mineral levels and maintaining increased bone mineral levels over a long period of time.

0108For example, about 1 month to about 12 months (for example, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, or about Over a treatment period of 11 months), one or more administrations of the formulations described herein may be performed. In some embodiments, the subject is administered one or more doses of the present formulation to maintain bone mineral levels. The term "maintaining bone mineral concentration" used here means that the increase in bone mineral concentration caused by the initial administration of this product is about 6 months, about 9 months, about 1 year, about 18 months, and about 2 years. , Or means that it does not decrease by more than about 1% to about 5% over the life of the patient. It will be appreciated that patients may require a separate treatment period to increase and maintain bone density.

0109In addition, it may be advantageous to administer multiple doses of the product, or to stagger the doses, depending on the treatment regimen selected for the particular subject. This product can be administered regularly for a period of 1 year or less (for example, 9 months or less, 6 months or less, or 3 months or less). In this regard, the product is about 7 days, or 2 weeks, or 3 weeks, or 1 month, or 5 weeks, or 6 weeks, or 7 weeks, or 2 months, or 9 weeks, or 10 weeks, or 11 weeks, or 3 months, or 13 weeks, or 14 weeks, or 15 weeks, or 4 months, or 17 weeks, or 18 weeks, or 19 weeks, or 5 months, or 21 weeks, or 22 weeks, or 23 weeks. , Or once every 6 or 12 months, can be administered to humans.

0110<u style="single">VII. Combination treatment</u> Treatment of a medical condition by combining two or more drugs that target the same pathogen or biochemical pathway is sometimes more effective than the single use of each drug at a therapeutically relevant dose. And results in reduced side effects. In some cases, the efficacy of the drug combination is additive (the effectiveness of the combination is approximately equal to the sum of the effects of each drug alone), but in other cases the effects are synergistic (the effectiveness of the combination is approximately equal to the sum of the effects of each drug alone). The effectiveness of the combination is greater than the sum of the effects of each drug given alone). The term "combination therapy" as used herein refers to a method in which two compounds are administered simultaneously, eg, in parallel, or one of the compounds is administered first, followed by a second agent. For example, it means that it can be delivered continuously. The desired result can be either subjective relief of one or more symptoms or objectively identifiable improvement in the recipient of the dose.

0111In some embodiments, the present formulation is administered with a standard therapeutic agent for the treatment of reduced bone mineral levels. As used herein, the term "standard treatment" generally refers to the treatment accepted by a clinician for a particular type of patient diagnosed with a type of disease. In some embodiments, standard therapeutic agents include reabsorption inhibitors, osteogenic agents, estrogen receptor antagonists (including, but not limited to, raloxifene, bazedoxifene, and lasofoxyfene), and osteoclasts. It is selected from the group consisting of drugs that have an irritating effect on the drug. In some embodiments, reabsorption inhibitors include, but are not limited to, bisphosphonates, but not limited to, alendronate, resedronate, ibandronate, and zoledronic acid, estrogen or estrogen analogs, selective estrogen receptor modulators. (SERM) and calcium sources, tibolone, calcitonin, calcitriol, and hormone replacement therapy include, but are not limited to. In some embodiments, the bone-forming agent includes parathyroid hormone (PTH) or a peptide fragment thereof, PTH-related protein (PTHrp), bone-forming protein, osteogenin, NaF, PGE.₂Agonists, statins, and RANK ligands (RANKL) include, but are not limited to. In some embodiments, the drug having an irritating effect on osteoclasts includes, but is not limited to, vitamin D, or a vitamin D derivative or mimic thereof.

0112In some embodiments, the formulation is administered to a subject if treatment with the standard therapeutic agents described herein is contraindicated. (Example)<u style="single">Example 1-Reduction of effective viscosity of sclerostin antibody preparation by calcium acetate</u> 10 mL of the selected anti-sclerostin antibody (75.7 mg / mL) was dialyzed against 2 liters of 10 mM Na (OAc) and 9% sucrose at 4 ° C for 2 hours. The selected anti-sclerostin antibody (75.7 mg / mL) was concentrated to about 160 mg / mL and diluted with water to about 140 mg / mL and 120 mg / mL. The absorbance of the diluted sample was determined to 120, 142, and 157 mg / mL, respectively.

011310 μL 1.0 M Ca (OAc)₂Was added to 1 mL of 120 mg / mL, 140 mg / mL, and 160 mg / mL samples. The absolute viscosity, pH, and osmolality of the sample were determined (see Table 2). Measure the absolute viscosity of a sample (500 μL) using a Brookfield LV-DVII cone plate viscometer with a CPE-40 spindle, where the temperature of a compatible sample cup is adjusted by a circulating water tank at a constant 25 ° C. did.

0114<tables num="2"><img id="000003" he="36" wi="136" file="JP6174176B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

0115The result is 10 mM Ca (OAc) added to the liquid composition of the selected antibody.₂Reduced the viscosity by about half. This experiment is performed for each of the antibodies Ab-4, Ab-5, Ab-13, Ab-14, Ab-19, Ab-20, and Ab-23.<u style="single">Example 2-Formulation adjustment</u> 10 mL of the selected anti-sclerostin antibody (75.7 mg / mL) was dialyzed against 2 liters of 10 mM Na (OAc), 6% sucrose or 4% sucrose at 4 ° C for 2 hours. Then, using Amicon, each sucrose preparation was concentrated to about 140 mg / mL and then diluted back with water to the target concentration (ie, 120 mg / mL, 140 mg / mL, and 160 mg / mL). The absorbance values of the diluted samples were determined to be 124 mg / mL (4% sucrose), 119.5 mg / mL (6% sucrose), 137.5 mg / mL (4% sucrose), and 142 mg / mL (6% sucrose), respectively. ..

011610 μL 1.0 M Ca (OAc)₂Was added to a 1 mL sample. The viscosity, osmolality, and pH of the sample were determined (see Table 3).<tables num="3"><img id="000004" he="57" wi="155" file="JP6174176B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

0117The assay was repeated as follows. 10 mL of the selected anti-sclerostin antibody (75.7 mg / mL) was dialyzed against 2 liters of 10 mM Na (OAc), 6% sucrose or 4% sucrose at 4 ° C for 2 hours. Each sucrose formulation was then concentrated to approximately 140 mg / mL using an Amicon filter and then diluted back with water to the target concentration (ie, 70 mg / mL, 100 mg / mL, and 120 mg / mL). The absorbance values of the diluted samples were 71 mg / mL (4% sucrose), 68.2 mg / mL (6% sucrose), 99.4 mg / mL (4% sucrose), 100.5 (6% sucrose), and 122 mg / mL (4), respectively. % Sucrose), and 113 mg / mL (6% sucrose).

0118The viscosity, osmolality, and pH of the sample were determined. See Table 4.<tables num="4"><img id="000005" he="115" wi="154" file="JP6174176B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

0119Ca (OAc)₂By lowering the pH of the buffer to 5.2, the pH of all final formulations was maintained at 5.25-5.307. The 4% sucrose preparation was below the isotonic range (250 to 350 mOsm / kg), while the 6% sucrose preparation was close to the median isotonic range.

012010 mM Ca (OAc) in reducing viscosity₂To further assess the effect of 6% sucrose containing, the above assay was repeated with additional concentrations of anti-sclerostin antibody up to 160 mg / mL.

0121Samples were prepared as described above at the following concentrations: 120 mg / mL, 140 mg / mL, and 160 mg / mL. 10 μL 1.0 M Ca (OAc)₂, PH 5.2 was added to each of the samples. The viscosity, osmolality, and pH of the sample were determined. See Table 5.

0122<tables num="5"><img id="000006" he="102" wi="154" file="JP6174176B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

0123The above experiments are performed for each of the antibodies Ab-4, Ab-5, Ab-13, Ab-14, Ab-19, Ab-20, and Ab-23.<u style="single">Example 3-Effect of calcium acetate in other high protein concentration preparations</u> In the following examples, it was determined whether calcium acetate reduces the viscosity of the preparation containing a high concentration of protein in addition to the sclerostin antibody.

0124Non-sclerostin antibodies # 1 to # 5 were determined to have concentrations of 131.6 mg / mL, 94 mg / mL, 113.2 mg / mL, 50 mg / mL, and 106.3, respectively. As used herein, the term "non-sclerostin antibody" means an antibody other than the sclerostin antibody described herein.

012510 μL 1.0 M Ca (OAc)₂Was added to the above 1 mL of 5 samples. The viscosity, pH, and osmolality of the sample were determined (see Table 6).

0126<tables num="6"><img id="000007" he="59" wi="139" file="JP6174176B2_D0001.tif" img-format="tif" img-content="drawing" /></tables>

0127Calcium acetate did not significantly reduce the viscosity of any of the samples.<u style="single">Example 4-Effect of non-calcium salt on viscosity of high-concentration anti-sclerostin antibody preparation</u> The following experiments were carried out to determine whether non-calcium salts could reduce the viscosity of the anti-sclerostin antibody preparation.

0128The selected anti-sclerostin antibody (same as Examples 1-2 above) was concentrated to about 130 mg / mL. 10 μL 1.0 M (NH₄)₂SO₄Or 1.0M DDL₄Was added to 1 mL of antibody sample. The viscosity of the control was determined to be 30 cP. DDL₄Was determined to significantly reduce the viscosity of the sample (DDL)₄+ Sample = 16cP). (NH₄)₂SO₄Did not significantly reduce the viscosity of the sample.<u style="single">Example 5-Effect of other calcium salts on the viscosity of high-concentration anti-sclerostin antibody preparation</u> The following experiments were carried out to determine whether calcium salts other than calcium acetate can reduce the viscosity of the anti-sclerostin antibody preparation.

0129The selected anti-sclerostin antibody (same as Examples 1-2 above) was concentrated to about 125 mg / mL. 10 μL of 25 mM CaCl₂Or 25 mM MgCl₂Was added to 1 mL of antibody sample. The viscosity of the control was determined to be 18.5 cP. CaCl₂And MgCl₂Was determined to significantly reduce the viscosity of the sample (CaCl)₂+ Sample = 9cP and MgCl₂+ Sample = 8).<u style="single">Example 6-Effect of calcium acetate on another anti-sclerostin antibody</u> The following experiment was carried out to determine whether calcium acetate is capable of reducing the viscosity of the anti-sclerostin antibody preparation, which comprises an anti-sclerostin antibody different from Examples 1 and 2 above.

0130The selected anti-sclerostin antibody was concentrated to about 131 mg / mL. 10 μL 1.0 M Ca (OAc)₂Was added to a 1 mL antibody sample. The viscosity of the control was determined to be 17.3 cP. Ca (OAc)₂Was determined to reduce the viscosity of the sample slightly (15.3 cP).

0131It is expected that many modifications and modifications in the practice of the present invention will be conceived by those skilled in the art by considering the currently preferred embodiments of the present invention. Therefore, the only limitation set on the scope of the invention is set forth in the appended claims.

0132All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referred to herein and / or listed in the application datasheet are by reference. The whole is incorporated herein.

0133Although specific embodiments of the invention have been described herein for illustrative purposes, it is understood from the above that various modifications may be made without departing from the spirit and scope of the invention. Will. The present invention also relates to: [1] A sterile liquid preparation having an absolute viscosity of about 10 cP or less, comprising (a) an anti-sclerostin immunoglobulin at a concentration of at least 70 mg / mL and (b) calcium acetate at a concentration in the range of about 1 mM to about 20 mM. [2] The immunoglobulins are SEQ ID NO: 14 and / or SEQ ID NO: 12, or SEQ ID NO: 86 and / or SEQ ID NO: 84, or SEQ ID NO: 68 and / or SEQ ID NO: 66, or SEQ ID NO: 154 and / or SEQ ID NO: 152, or SEQ ID NO: Containing the amino acid sequences of SEQ ID NO: 182 and / or SEQ ID NO: 180, or SEQ ID NO: 208 and / or SEQ ID NO: 207, or SEQ ID NO: 216 and / or SEQ ID NO: 214, or SEQ ID NO: 238 and / or SEQ ID NO: 236, [1] The formulation described in. [3] (c) The preparation according to [1], further comprising an acetate buffer having a concentration of about 5 mM to about 15 mM, for example, sodium acetate. [Four] Approximately containing (a) anti-sclerostin immunoglobulins at concentrations from about 70 mg / mL to about 200 mg / mL and (b) acetates and / or acetate buffers at concentrations ranging from about 10 mM to about 50 mM acetates. A sterile liquid formulation having an absolute viscosity of 10 cP or less, wherein the immunoglobulin is SEQ ID NO: 14 and / or SEQ ID NO: 12, or SEQ ID NO: 86 and / or SEQ ID NO: 84, or SEQ ID NO: 68 and / or SEQ ID NO. 66, or SEQ ID NO: 154 and / or SEQ ID NO: 152, or SEQ ID NO: 182 and / or SEQ ID NO: 180, or SEQ ID NO: 208 and / or SEQ ID NO: 207, or SEQ ID NO: 216 and / or SEQ ID NO: 214, or SEQ ID NO: 238. And / or a sterile liquid formulation comprising the amino acid sequence of SEQ ID NO: 236. [Five] The preparation according to any one of [1] to [4], which has a total osmolality of less than about 350 mOsm / L. [6] The preparation according to any one of [1] to [5], wherein the immunoglobulin is present at a concentration of at least 120 mg / mL, or optionally 140 mg / mL. [7] The preparation according to any one of [1] to [6], wherein the absolute viscosity of the preparation is about 8 cP or less, or optionally about 6 cP or less. [8] The preparation according to any one of [1] to [7], wherein the preparation further contains a polyol such as sucrose in an amount in the range of about 4% w / v to about 6% w / v. [9] The preparation according to any one of [1] to [8], wherein the preparation has a pH in the range of about 4.5 to about 6, or optionally about 5 to about 5.5. [Ten] A method for reducing the viscosity of a protein preparation, which comprises adding calcium acetate at a concentration of about 1 mM to about 20 mM to an anti-sclerostin immunoglobulin preparation, wherein the preparation is from about 70 mg / mL to about 200 mg /. A method, wherein the viscosity of the formulation containing mL of immunoglobulin and containing calcium acetate is reduced as compared to the viscosity of the antibody formulation containing no calcium acetate. [11] The method of [10], wherein the immunoglobulin comprises the amino acid sequence of SEQ ID NO: 86 and / or SEQ ID NO: 84. [12] (a) Ab-5 with a concentration of at least 70 mg / mL to about 200 mg / mL, (b) calcium acetate with a concentration in the range of about 1 mM to about 20 mM, and (c) about 4% w / v to about 6%. A sterile liquid formulation having an absolute viscosity of about 10 cP or less, which comprises an amount in the range of w / v, eg, with a polyol such as sucrose. [13] Sterilized liquid preparation (a) Anti-sclerostin immunoglobulins at a concentration of at least 70 mg / mL to about 200 mg / mL, SEQ ID NO: 14 and / or SEQ ID NO: 12, or SEQ ID NO: 86 and / or SEQ ID NO: 84, or SEQ ID NO: 68 and /. Alternatively, SEQ ID NO: 66, or SEQ ID NO: 154 and / or SEQ ID NO: 152, or SEQ ID NO: 182 and / or SEQ ID NO: 180, or SEQ ID NO: 208 and / or SEQ ID NO: 207, or SEQ ID NO: 216 and / or SEQ ID NO: 214, or An immunoglobulin comprising the amino acid sequences of SEQ ID NO: 220 and / or SEQ ID NO: 218, or SEQ ID NO: 238 and / or SEQ ID NO: 236. (b) Calcium acetate having a concentration in the range of about 1 mM to about 20 mM, which reduces the absolute viscosity of the preparation by at least 10% as compared with the viscosity of the antibody preparation containing no calcium acetate. Contains sterile liquid formulations. [14] To treat patients undergoing orthopedic surgery, dental surgery, implant surgery, joint replacement, bone grafting, bone cosmetic surgery, and bone repair such as fracture healing, non-adhesive healing, protracted healing, and facial reconstruction. A method of using a therapeutically effective amount of the preparation according to any one of [1] to [9], [12] and [13].