Lyophilized ENPP1 polypeptide formulations and uses thereof

Abstract

In certain aspects, the present invention provides novel lyophilized formulations of ENPP1 polypeptides and methods for treating indications associated with ENPP1 deficiency using such lyophilized and reconstituted formulations of ENPP1. The formulations and methods provided herein are useful in treating diseases associated with ENPP1 deficiency, such as pathological calcification or pathological ossification.

Description

[Technical field]

[0001] cross reference This application claims priority to U.S. Provisional Patent Application No. 63 / 248,303, filed September 24, 2021, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in xml. format and is incorporated herein by reference in its entirety. The xml. copy, created on September 23, 2022, is titled 4427-11902_sequence.txt and is 137KB in size. [Background technology]

[0003] The human ectonucleotide pyrophosphatase (ENPP) protein family includes seven extracellular glycosylated proteins (i.e., ENPP1-ENPP7) that hydrolyze phosphodiester bonds. ENPPs are cell surface enzymes, with the exception of ENPP2, which is exported to the plasma membrane and cleaved by furin and released into the extracellular fluid. ENPP enzymes share a high degree of sequence and structural homology but display diverse substrate specificities ranging from nucleotides to lipids.

[0004] ENPP1 (also known as PC-1) and ENPP3 are type 2 extracellular membrane-bound glycoproteins located on mineralizing matrix vesicles of osteoblasts and chondrocytes that hydrolyze extracellular nucleotides (mainly ATP) to adenosine monophosphate (AMP) and inorganic pyrophosphate (PPi). PPi functions as a potent inhibitor of ectopic tissue mineralization by binding to nascent hydroxyapatite (HA) crystals, thereby preventing the further growth of these crystals. ENPP1 generates PPi via hydrolysis of nucleotide triphosphates (NTPs).

[0005] Ectopic tissue calcification is associated with a number of human diseases, including chronic joint disease and acute lethal neonatal syndrome. To prevent unwanted tissue calcification, factors that promote and inhibit tissue calcification must be kept in tight balance. The balance of extracellular inorganic pyrophosphate (PPi) and phosphate (Pi) is a key regulator of ectopic tissue calcification.

[0006] Summary of the Invention The present disclosure features, inter alia, a lyophilized formulation comprising an ENPP1 polypeptide. Such formulations feature, inter alia, remarkable stability over a wide range of temperatures. Thus, in one aspect, the present disclosure features a lyophilized polypeptide formulation comprising an ENPP1 polypeptide.

[0007] In some embodiments, the formulations described herein include a buffering agent. In some embodiments, the buffering agent maintains a pH range of pH 6-7 when reconstituted in solution. In some embodiments, the buffering agent maintains a pH range of pH 7-8 when reconstituted in solution. In some embodiments, the buffering agent maintains a pH range of pH 6-8 when reconstituted in solution. In some embodiments, the buffering agent is selected from the group consisting of succinate, citrate, bicarbonate, phosphate, Tris, or glycylglycine. In some embodiments, the buffering agent is succinate, citrate, or phosphate. In some embodiments, the buffering agent is citrate. In some embodiments, the buffering agent is succinate. In some embodiments, the buffering agent is phosphate. In some embodiments, the buffering agent is in a concentration range of 5 mM to 100 mM when reconstituted in solution. In some embodiments, the buffering agent increases the onset temperature of aggregate formation. In some embodiments, the buffering agent increases the onset temperature of aggregate formation by at least 2° C. In some embodiments, the buffering agent reduces the formation of high molecular weight species. In some embodiments, the buffer reduces the formation of high molecular weight species by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%.

[0008] In some embodiments, the formulations described herein include one or more pharma- ceutically acceptable additives. In some embodiments, such pharma-ceutically acceptable additives are or include stabilizers, amino acids, salts, metal ions, and surfactants. In some embodiments, the stabilizer is or includes a sugar, carbohydrate, or polysaccharide. In some embodiments, such sugars are selected from the group consisting of sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, or sorbitol. In some embodiments, the one or more pharma-ceutically acceptable additives are or include mannitol. In some embodiments, the pharma-ceutically acceptable additives are or include sucrose. In some embodiments, the pharma-ceutically acceptable additives are or include an amino acid. In some embodiments, such amino acids are selected from the group consisting of glycine, arginine, histidine, alanine, proline, serine, and glutamic acid. In some embodiments, the amino acid is arginine. In some embodiments, the pharma- ceutically acceptable excipient is a salt, such as, but not limited to, sodium chloride (NaCl), calcium chloride (CaCl2). 、The salt is zinc chloride (ZnCl2) and / or magnesium chloride (MgCl2). In some embodiments, the salt is calcium chloride (CaCl2). In some embodiments, the pharma- ceutically acceptable excipient is a surfactant, such as, but not limited to, polysorbates, poloxamers, tritons, sodium dodecyl sulfate, sodium lauryl sulfate, sodium octyl glucoside, lauryl sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroamidopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, paclitaxel, cyclohexyl-betaine ... The surfactant may be selected from the group consisting of polysorbate 20 (PS20), polysorbate 80 (PS80), or polysorbate 188 (PS188). In some embodiments, the surfactant is polysorbate 20 (PS20).

[0009] In some embodiments, the formulations described herein comprise a buffer, a stabilizer, a salt, an amino acid, and a surfactant. In some embodiments, the buffer is citrate or succinate, the stabilizer is sucrose or mannitol, the salt is calcium chloride (CaCl2), and the surfactant is polysorbate 20 (PS20).

[0010] In some embodiments, the pH of the formulations described herein (e.g., for lyophilized formulations, when reconstituted in a reconstitution solution or sterile water) is between pH 6.2 and pH 6.5. In some embodiments, the pH of the formulations described herein (e.g., for lyophilized formulations, when reconstituted in a reconstitution solution or sterile water) is between about pH 6.0 and about pH 7.0, inclusive.

[0011] In some embodiments, the formulations described herein contain about 15 to about 25 mM citrate.

[0012] In some embodiments, the formulations described herein comprise about 75 mM to about 95 mM each of sucrose, mannitol, or a combination thereof.

[0013] In some embodiments, the formulations described herein contain about 100 mM to about 300 mM sucrose.

[0014] In some embodiments, the formulations described herein contain about 1 mM to about 3 mM calcium chloride.

[0015] In some embodiments, the formulations include a surfactant. The surfactant can be or include, for example, polysorbate 20 (PS20), polysorbate 80 (PS80), or poloxamer Px188. In some embodiments, the surfactant is or includes PS20. In some embodiments, the surfactant is present at a concentration ranging from about 0.02% to about 0.10% (w / v) when reconstituted in solution. In some embodiments, the formulations described herein include about 0.005% to about 0.1% (w / v) of a surfactant. In some embodiments, the formulations described herein include about 0.005% to about 0.1% (w / v) of PS20. In some embodiments, the surfactant increases resistance to physical stress.

[0016] In some embodiments, the pharma- ceutically acceptable excipient(s), when reconstituted in solution (e.g., with reconstitution buffer or sterile water), has a concentration ranging from about 5 mM to about 300 mM.

[0017] In some embodiments, the one or more pharma- ceutically acceptable excipients reduce the formation of high molecular weight species.

[0018] In some embodiments, the one or more pharma- ceutically acceptable excipients reduce the formation of high molecular weight species by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%.

[0019] In some embodiments, the formulation includes an ENPP1 polypeptide cofactor, such as, but not limited to, calcium, zinc, and / or adenosine monophosphate. In some embodiments, the ENPP1 polypeptide cofactor is CaCl2, CaSO4, ZnCl2, ZnSO4, and / or adenosine monophosphate. In some embodiments, the ENPP1 polypeptide cofactor is CaCl2 and / or adenosine monophosphate. In some embodiments, the ENPP1 polypeptide cofactor is present at a concentration ranging from about 1 mM to about 10 mM when reconstituted in solution. In some embodiments, the ENPP1 polypeptide cofactor reduces the formation of high molecular weight species by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%.

[0020] In some embodiments, the formulations described herein are homogeneous in particle size or are characterized as being homogeneous in particle size. In some embodiments, the formulations include a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold reduction in larger particle size as determined by microflow imaging analysis.

[0021] In some embodiments of any of the formulations described herein, the ENPP1 polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence comprising SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0022] In some embodiments, the ENPP1 polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence comprising SEQ ID NO:2.

[0023] In some embodiments, the ENPP1 polypeptide is a fusion protein comprising a soluble ENPP1 polypeptide domain and one or more heterologous protein moieties. In some embodiments, the heterologous protein moieties increase the circulating half-life of the soluble ENPP1 polypeptide in a mammal. In some embodiments, the heterologous protein moiety comprises an Fc domain. In some embodiments, the Fc domain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the ENPP1 polypeptide is an ENPP1-Fc fusion protein. In some embodiments, the ENPP1 polypeptide further comprises a heterologous moiety. In some embodiments, the heterologous moiety is selected from the group consisting of a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, and a lipid moiety.

[0024] In some embodiments, the ENPP1 polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence comprising SEQ ID NO: 13. In some embodiments, the ENPP1 polypeptide is at least 90% identical to SEQ ID NO: 13. In some embodiments, the ENPP1 polypeptide is at least 95% identical to SEQ ID NO: 13. In some embodiments, the ENPP1 polypeptide is at least 99% identical to SEQ ID NO: 13. In some embodiments, the ENPP1 polypeptide comprises SEQ ID NO: 13. In some embodiments, the ENPP1 polypeptide consists of SEQ ID NO: 13. In some embodiments, the ENPP1 polypeptide comprises an Fc domain. The Fc domain may comprise or consist of the amino acid sequence depicted in SEQ ID NO: 12. In some embodiments, the ENPP1 polypeptide comprises a linker amino acid sequence joining the ENPP1 polypeptide moiety and the Fc domain. In some embodiments, the linker amino acid sequence comprises or consists of the amino acid sequence LIN. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:9. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:10. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:11.

[0025] In some embodiments, the ENPP1 polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence comprising SEQ ID NO: 14. In some embodiments, the ENPP1 polypeptide is at least 90% identical to SEQ ID NO: 14. In some embodiments, the ENPP1 polypeptide is at least 95% identical to SEQ ID NO: 14. In some embodiments, the ENPP1 polypeptide is at least 99% identical to SEQ ID NO: 14. In some embodiments, the ENPP1 polypeptide comprises SEQ ID NO: 14. In some embodiments, the ENPP1 polypeptide consists of SEQ ID NO: 14. In some embodiments, the ENPP1 polypeptide comprises an Fc domain. The Fc domain may comprise or consist of the amino acid sequence depicted in SEQ ID NO: 12. In some embodiments, the ENPP1 polypeptide comprises a linker amino acid sequence joining the ENPP1 polypeptide moiety and the Fc domain. In some embodiments, the linker amino acid sequence comprises or consists of the amino acid sequence LIN. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:9. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:10. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:11.

[0026] In some embodiments, the ENPP1 polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence comprising SEQ ID NO: 15. In some embodiments, the ENPP1 polypeptide is at least 90% identical to SEQ ID NO: 15. In some embodiments, the ENPP1 polypeptide is at least 95% identical to SEQ ID NO: 15. In some embodiments, the ENPP1 polypeptide is at least 99% identical to SEQ ID NO: 15. In some embodiments, the ENPP1 polypeptide comprises SEQ ID NO: 15. In some embodiments, the ENPP1 polypeptide consists of SEQ ID NO: 15. In some embodiments, the ENPP1 polypeptide comprises an Fc domain. The Fc domain may comprise or consist of the amino acid sequence depicted in SEQ ID NO: 12. In some embodiments, the ENPP1 polypeptide comprises a linker amino acid sequence joining the ENPP1 polypeptide moiety and the Fc domain. In some embodiments, the linker amino acid sequence comprises or consists of the amino acid sequence LIN. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:9. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:10. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:11.

[0027] In some embodiments, the formulation comprises an ENPP1 polypeptide, a buffer, a pharma- ceutically acceptable salt, a stabilizer, a surfactant, and one or more pharma- ceutically acceptable additives. In some embodiments, the buffer is a citrate buffer. The citrate buffer can be, for example, trisodium citrate dihydrate. In some embodiments, the pharma- cetically acceptable salt is calcium chloride. In some embodiments, the stabilizer is a sugar, such as sucrose. In some embodiments, the one or more pharma- cetically acceptable additives are or comprise mannitol. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 13. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 14. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 15. In some embodiments, the ENPP1 polypeptide comprises an Fc domain. The Fc domain can comprise or consist of the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the ENPP1 polypeptide comprises a linker amino acid sequence joining the ENPP1 polypeptide moiety and the Fc domain. In some embodiments, the linker amino acid sequence comprises or consists of the amino acid sequence LIN. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:9. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:10. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:11.

[0028] In some embodiments, the formulation comprises an ENPP1 polypeptide, a citrate buffer, calcium chloride, sucrose, and polysorbate. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 13. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 14. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 15. In some embodiments, the ENPP1 polypeptide comprises an Fc domain. The Fc domain may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the ENPP1 polypeptide comprises a linker amino acid sequence joining the ENPP1 polypeptide moiety and the Fc domain. In some embodiments, the linker amino acid sequence comprises or consists of the amino acid sequence LIN. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO: 9. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO: 10. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:11.

[0029] In some embodiments, the formulation comprises an ENPP1 polypeptide, trisodium citrate dihydrate, calcium chloride dihydrate, sucrose, and polysorbate 20. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 13. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 14. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 15. In some embodiments, the ENPP1 polypeptide comprises an Fc domain. The Fc domain may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the ENPP1 polypeptide comprises a linker amino acid sequence joining the ENPP1 polypeptide moiety and the Fc domain. In some embodiments, the linker amino acid sequence comprises or consists of the amino acid sequence LIN. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO: 9. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO: 10. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:11.

[0030] In some embodiments, the formulation comprises an ENPP1 polypeptide, a citrate buffer, calcium chloride, sucrose, mannitol, and polysorbate. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 13. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 14. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 15. In some embodiments, the ENPP1 polypeptide comprises an Fc domain. The Fc domain may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the ENPP1 polypeptide comprises a linker amino acid sequence joining the ENPP1 polypeptide moiety and the Fc domain. In some embodiments, the linker amino acid sequence comprises or consists of the amino acid sequence LIN. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO: 9. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO: 10. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:11.

[0031] In some embodiments, the formulation comprises an ENPP1 polypeptide, trisodium citrate dihydrate, calcium chloride dihydrate, sucrose, D(-) mannitol, and polysorbate 20. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 13. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 14. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 15. In some embodiments, the ENPP1 polypeptide comprises an Fc domain. The Fc domain may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the ENPP1 polypeptide comprises a linker amino acid sequence joining the ENPP1 polypeptide moiety and the Fc domain. In some embodiments, the linker amino acid sequence comprises or consists of the amino acid sequence LIN. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO: 9. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO: 10. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:11.

[0032] In some embodiments, the formulation comprises, per 0.5 ml final reconstituted volume, about 25 mg of ENPP1 polypeptide, about 2.94 mg of trisodium citrate dihydrate, about 0.15 mg of calcium chloride dihydrate, about 30 mg of sucrose, about 7.5 mg of D(-) mannitol, and about 0.25 mg of polysorbate (e.g., PS20). In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 13. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 14. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 15. In some embodiments, the ENPP1 polypeptide comprises an Fc domain. The Fc domain comprises or consists of the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the ENPP1 polypeptide comprises a linker amino acid sequence joining the ENPP1 polypeptide moiety and the Fc domain. In some embodiments, the linker amino acid sequence comprises or consists of the amino acid sequence LIN. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:9. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:10. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:11.

[0033] In some embodiments, the formulation may be reconstituted in a sterile injection solution. In some embodiments, the formulation may be reconstituted in a reconstitution solution or sterile water. In some embodiments, the reconstitution solution comprises a pharma- ceutically acceptable carrier and / or additive. In some embodiments, the pharma- ceutically acceptable carrier is selected from saline, purified water, or sterile water for injection. In some embodiments, the formulation is completely reconstituted within a period of less than 100 seconds, 80 seconds, 70 seconds, 68 seconds, 65 seconds, or 60 seconds.

[0034] In some embodiments, the reconstituted formulation comprises at least or about 50 mg / mL ENPP1 polypeptide, about 20 mM citrate at about pH 6.3, about 2 mM calcium chloride, about 175 mM sucrose, about 82 mM mannitol, and about 0.05% w / v polysorbate. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 13. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 14. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 15. In some embodiments, the ENPP1 polypeptide comprises an Fc domain. The Fc domain may comprise or consist of the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the ENPP1 polypeptide comprises a linker amino acid sequence joining the ENPP1 polypeptide moiety and the Fc domain. In some embodiments, the linker amino acid sequence comprises or consists of the amino acid sequence LIN. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

[0035] In some embodiments, the reconstituted formulation comprises at least or about 50 mg / mL ENPP1 polypeptide, about 20 mM citrate at about pH 6.3, about 2 mM calcium chloride, about 175 mM sucrose, about 82 mM (D) mannitol, and about 0.05% w / v polysorbate 20. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 13. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 14. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 15. In some embodiments, the ENPP1 polypeptide comprises an Fc domain. The Fc domain may comprise or consist of the amino acid sequence shown in SEQ ID NO: 12. In some embodiments, the ENPP1 polypeptide comprises a linker amino acid sequence joining the ENPP1 polypeptide moiety and the Fc domain. In some embodiments, the linker amino acid sequence comprises or consists of the amino acid sequence LIN. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

[0036] In some embodiments, the reconstituted formulation comprises at least or about 50 mg / mL ENPP1 polypeptide, about 20 mM citrate at about pH 6.3, about 88 mM sucrose, about 82 mM mannitol, about 2 mM calcium chloride, and about 0.05% w / v polysorbate 20. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 13. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 14. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 15. In some embodiments, the ENPP1 polypeptide comprises an Fc domain. The Fc domain may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the ENPP1 polypeptide comprises a linker amino acid sequence joining the ENPP1 polypeptide moiety and the Fc domain. In some embodiments, the linker amino acid sequence comprises or consists of the amino acid sequence LIN. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

[0037] In some embodiments, the reconstituted formulation comprises at least or about 50 mg / mL ENPP1 polypeptide, about 20 mM citrate at about pH 6.3, about 263 mM sucrose, about 2 mM calcium chloride, and about 0.05% w / v polysorbate 20. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 13. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 14. In some embodiments, the ENPP1 polypeptide comprises or consists of SEQ ID NO: 15. In some embodiments, the ENPP1 polypeptide comprises an Fc domain. The Fc domain may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 12. In some embodiments, the ENPP1 polypeptide comprises a linker amino acid sequence joining the ENPP1 polypeptide moiety and the Fc domain. In some embodiments, the linker amino acid sequence comprises or consists of the amino acid sequence LIN. In some embodiments, the ENPP1 polypeptide consists of or comprises SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0038] In some embodiments, the lyophilized formulation exhibits long-term stability at -80° C. to 40° C. In some embodiments, the lyophilized formulation has a shelf life of at least 3, 6, 12, 24, 36, 48, or 60 months. In some embodiments, the reconstituted formulation has a shelf life of at least 1, 2, 3, 4, 5, 6, 12, 18, 24, 48, or 60 hours.

[0039] In some embodiments, the lyophilized formulation is shelf stable for at least 36 months when stored at 5° C. In some embodiments, the formulation is shelf stable for 48 months when stored at 5° C. The formulation is shelf stable for at least 24 months when stored at room temperature (RT-25° C.). The formulation is shelf stable for at least 36 months when stored at room temperature. The formulation is shelf stable for 48 months when stored at room temperature. The formulation is shelf stable for at least 6 months when stored at 40° C. The formulation is shelf stable for 12 months when stored at 40° C.

[0040] In some embodiments, the reconstituted formulation is stable at a concentration ranging from 1 mg / ml to 50 mg / ml for at least 8 hours at room temperature, In some embodiments, the reconstituted formulation is stable at a concentration ranging from 1 mg / ml to 50 mg / ml for at least 24 hours at 5° C.

[0041] In some embodiments, the reconstituted formulation is stable at a concentration ranging from 1 mg / ml to 10 mg / ml for at least 12 hours at 5° C. In some embodiments, the reconstituted formulation is stable at 1 mg / ml for at least 12 hours at 5° C. In some embodiments, the reconstituted formulation is stable at 2.5 mg / ml for at least 12 hours at 5° C. In some embodiments, the reconstituted formulation is stable at 10 mg / ml for at least 12 hours at 5° C.

[0042] In some embodiments, the reconstituted formulation is stored in a vial. In some embodiments, the reconstituted formulation is stored in a syringe. In some embodiments, the reconstituted formulation is stable for at least 12 hours when stored in a vial at 5° C. In some embodiments, the reconstituted formulation is stable for at least 8 hours when stored in a syringe at 5° C.

[0043] Accordingly, the disclosure also features a vial containing a lyophilized formulation comprising ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, the vial containing 1-100 mg, e.g., 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, or 50 mg, or 100 mg of ENPP1 or ENPP1-Fc.

[0044] In some embodiments, the vial contains a lyophilized formulation comprising ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 100 mg of ENPP1 or ENPP1-Fc.

[0045] In some embodiments, the vial contains a lyophilized formulation comprising ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 50 mg of ENPP1 or ENPP1-Fc.

[0046] In some embodiments, the vial contains a lyophilized formulation comprising ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 25 mg of ENPP1 or ENPP1-Fc.

[0047] In some embodiments, the vial contains a lyophilized formulation comprising ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 10 mg of ENPP1 or ENPP1-Fc.

[0048] In some embodiments, the vial contains a lyophilized formulation comprising ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 5 mg of ENPP1 or ENPP1-Fc.

[0049] In some embodiments, the vial contains a lyophilized formulation comprising ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 2.5 mg of ENPP1 or ENPP1-Fc.

[0050] In some embodiments, the vial contains a lyophilized formulation comprising ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 1 mg of ENPP1 or ENPP1-Fc.

[0051] Accordingly, the disclosure also features a vial containing a formulation comprising reconstituted ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, the vial containing 0.1 mg / ml to 100 mg / ml, e.g., 0.1 mg / ml, 1 mg / ml, 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, or 50 mg / ml, or 100 mg / ml of ENPP1 or ENPP1-Fc.

[0052] In some embodiments, the vial contains a formulation comprising reconstituted ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 100 mg / ml of ENPP1 or ENPP1-Fc.

[0053] In some embodiments, the vial contains a formulation comprising reconstituted ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, wherein the vial contains 50 mg / ml of ENPP1 or ENPP1-Fc.

[0054] In some embodiments, the vial contains a formulation comprising reconstituted ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, wherein the vial contains 25 mg / ml of ENPP1 or ENPP1-Fc.

[0055] In some embodiments, the vial contains a formulation comprising reconstituted ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 10 mg / ml of ENPP1 or ENPP1-Fc.

[0056] In some embodiments, the vial contains a formulation comprising reconstituted ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 5 mg / ml of ENPP1 or ENPP1-Fc.

[0057] In some embodiments, the vial contains a formulation comprising reconstituted ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, wherein the vial contains 2.5 mg / ml of ENPP1 or ENPP1-Fc.

[0058] In some embodiments, the vial contains a formulation comprising reconstituted ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 1 mg / ml of ENPP1 or ENPP1-Fc.

[0059] In some embodiments, the vial contains a formulation comprising reconstituted ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, wherein the vial contains 0.5 mg / ml of ENPP1 or ENPP1-Fc.

[0060] In some embodiments, the vial contains a formulation comprising reconstituted ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, wherein the vial contains 0.1 mg / ml of ENPP1 or ENPP1-Fc.

[0061] Accordingly, the disclosure also features a syringe containing a lyophilized formulation comprising ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, wherein the vial contains 0.1-100 mg, e.g., 0.1 mg, 1 mg, 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, or 50 mg, or 100 mg of ENPP1 or ENPP1-Fc.

[0062] In some embodiments, the syringe contains a lyophilized formulation comprising ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 100 mg of ENPP1 or ENPP1-Fc.

[0063] In some embodiments, the syringe contains a lyophilized formulation comprising ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 50 mg of ENPP1 or ENPP1-Fc.

[0064] In some embodiments, the syringe contains a lyophilized formulation comprising ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 25 mg of ENPP1 or ENPP1-Fc.

[0065] In some embodiments, the syringe contains a lyophilized formulation comprising ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 10 mg of ENPP1 or ENPP1-Fc.

[0066] In some embodiments, the syringe contains a lyophilized formulation comprising ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 5 mg of ENPP1 or ENPP1-Fc.

[0067] In some embodiments, the syringe contains a lyophilized formulation comprising ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 2.5 mg of ENPP1 or ENPP1-Fc.

[0068] In some embodiments, the syringe contains a lyophilized formulation comprising ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 1 mg of ENPP1 or ENPP1-Fc.

[0069] In some embodiments, the syringe contains a lyophilized formulation comprising ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 0.5 mg of ENPP1 or ENPP1-Fc.

[0070] In some embodiments, the syringe contains a lyophilized formulation comprising ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 0.1 mg of ENPP1 or ENPP1-Fc.

[0071] Accordingly, the disclosure also features a syringe containing a formulation comprising reconstituted ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, wherein the vial contains 0.1 mg / ml to 100 mg / ml, e.g., 0.1 mg / ml, 1 mg / ml, 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, or 50 mg / ml, or 100 mg / ml of ENPP1 or ENPP1-Fc.

[0072] In some embodiments, the syringe contains a formulation comprising reconstituted ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 100 mg / ml of ENPP1 or ENPP1-Fc.

[0073] In some embodiments, the syringe contains a formulation comprising reconstituted ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 50 mg / ml of ENPP1 or ENPP1-Fc.

[0074] In some embodiments, the syringe contains a formulation comprising reconstituted ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 25 mg / ml of ENPP1 or ENPP1-Fc.

[0075] In some embodiments, the syringe contains a formulation comprising reconstituted ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 10 mg / ml of ENPP1 or ENPP1-Fc.

[0076] In some embodiments, the syringe contains a formulation comprising reconstituted ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 5 mg / ml of ENPP1 or ENPP1-Fc.

[0077] In some embodiments, the syringe contains a formulation comprising reconstituted ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 2.5 mg / ml of ENPP1 or ENPP1-Fc.

[0078] In some embodiments, the syringe contains a formulation comprising reconstituted ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 1 mg / ml of ENPP1 or ENPP1-Fc.

[0079] In some embodiments, the syringe contains a formulation comprising reconstituted ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 0.5 mg / ml of ENPP1 or ENPP1-Fc.

[0080] In some embodiments, the syringe contains a formulation comprising reconstituted ENPP1, preferably ENPP1-Fc, and one or more of a buffer, bulking agent, stabilizer, and / or surfactant, and the vial contains 0.1 mg / ml of ENPP1 or ENPP1-Fc.

[0081] In some embodiments, the lyophilized ENPP1, preferably ENPP1-Fc polypeptide is stored in a vial. In some embodiments, the vial contains 0.1 mg of lyophilized ENPP1, preferably ENPP1-Fc polypeptide. In some embodiments, the vial contains 0.5 mg of lyophilized ENPP1, preferably ENPP1-Fc polypeptide. In some embodiments, the vial contains 1 mg of lyophilized ENPP1, preferably ENPP1-Fc polypeptide. In some embodiments, the vial contains 5 mg of lyophilized ENPP1, preferably ENPP1-Fc polypeptide. In some embodiments, the vial contains 10 mg of lyophilized ENPP1, preferably ENPP1-Fc polypeptide. In some embodiments, the vial contains 15 mg of lyophilized ENPP1, preferably ENPP1-Fc polypeptide. In some embodiments, the vial contains 25 mg of lyophilized ENPP1, preferably ENPP1-Fc polypeptide. In another embodiment, the vial contains 50 mg of lyophilized ENPP1, preferably ENPP1-Fc polypeptide.In another embodiment, the vial contains 100 mg of lyophilized ENPP1, preferably ENPP1-Fc polypeptide.

[0082] In some embodiments, the lyophilized ENPP1-Fc formulation is reconstituted for administration and stored in a syringe. In some embodiments, the syringe contains 100 mg / ml ENPP1-Fc for administration or for subsequent dilution. In some embodiments, the syringe contains 50 mg / ml ENPP1-Fc for administration or for subsequent dilution. In some embodiments, the syringe contains 25 mg / ml ENPP1-Fc for administration or for subsequent dilution. In some embodiments, the syringe contains 20 mg / ml ENPP1-Fc for administration or for subsequent dilution. In some embodiments, the syringe contains 15 mg / ml ENPP1-Fc for administration or for subsequent dilution. In some embodiments, the syringe contains 10 mg / ml ENPP1-Fc for administration or for subsequent dilution. In some embodiments, the syringe contains 5 mg / ml ENPP1-Fc for administration or for subsequent dilution. In some embodiments, the syringe contains 4 mg / ml ENPP1-Fc for administration or for subsequent dilution. In some embodiments, the syringe contains 3 mg / ml ENPP1-Fc for administration or for subsequent dilution. In some embodiments, the syringe contains 2.5 mg / ml ENPP1-Fc for administration or for subsequent dilution. In some embodiments, the syringe contains 2 mg / ml ENPP1-Fc for administration or for subsequent dilution. In some embodiments, the syringe contains 1 mg / ml ENPP1-Fc for administration or for subsequent dilution. In some embodiments, the syringe contains 0.5 mg / ml ENPP1-Fc for administration or for subsequent dilution. In some embodiments, the syringe contains 0.1 mg / ml ENPP1-Fc for administration or for subsequent dilution.

[0083] In some embodiments, the ENPP1-Fc formulation is administered to adults at a dose ranging from 0.2 mg / kg to 1.8 mg / ml. In some embodiments, the ENPP1-Fc formulation is administered to children and infants weighing more than 1 kg at a dose ranging from 0.2 mg / kg to 0.6 mg / kg. In some embodiments, the ENPP1-Fc formulation is administered to low birth weight infants weighing less than 1 kg at a dose ranging from 0.05 mg / kg to 0.2 mg / kg. In some embodiments, the ENPP1-Fc formulation is administered to infants weighing more than 1 kg at a dose ranging from 0.2 mg / kg. In some embodiments, the ENPP1-Fc formulation is administered to low birth weight infants weighing less than 1 kg at a dose of 0.1 mg / kg.

[0084] In some embodiments, any of the lyophilized formulations described herein comprises a therapeutically effective dose.

[0085] In some embodiments, any of the reconstituted formulations described herein comprises a therapeutically effective dose.

[0086] In yet another aspect, the disclosure features a method for producing or making any of the lyophilized formulations described herein. The method includes mixing an ENPP1 polypeptide (such as a fusion polypeptide including the catalytic domain of ENPP1 and an Fc region of an immunoglobulin) with one or more of a buffer, a stabilizer, a salt, an amino acid, and a surfactant. In some embodiments, the method includes mixing an ENPP1 polypeptide with a citrate buffer, sucrose, mannitol, calcium chloride, and polysorbate 20.

[0087] In yet another aspect, the disclosure features a method for producing a pharmaceutical solution comprising an ENPP1 polypeptide. The method includes contacting any of the lyophilized polypeptide formulations described herein with a sterile reconstitution solution or sterile water, thereby producing a reconstituted solution comprising an ENPP1 polypeptide. In some embodiments, the reconstitution solution includes a pharma- ceutically acceptable carrier and / or additive. In some embodiments, the pharma- ceutically acceptable carrier is selected from saline, purified water, or sterile water for injection. In some embodiments, the formulation is completely reconstituted within a period of less than 100 seconds, 80 seconds, 70 seconds, 68 seconds, 65 seconds, or 60 seconds. In some embodiments, the reconstituted formulation includes at least or about 50 mg / mL ENPP1 polypeptide, about 20 mM citrate at about pH 6.3, about 2 mM calcium chloride, about 175 mM sucrose, about 82 mM mannitol, and about 0.05% w / v polysorbate. In some embodiments, the reconstituted formulation comprises at least or about 50 mg / mL ENPP1 polypeptide, about 20 mM citrate at about pH 6.3, about 2 mM calcium chloride, about 175 mM sucrose, about 82 mM (D) mannitol, and about 0.05% w / v polysorbate 20. In some embodiments, the reconstituted formulation comprises at least or about 50 mg / mL ENPP1 polypeptide, about 20 mM citrate at about pH 6.3, about 88 mM sucrose, about 82 mM mannitol, about 2 mM calcium chloride, and about 0.05% w / v polysorbate 20. In some embodiments, the reconstituted formulation contains at least or about 50 mg / mL of ENPP1 polypeptide, about 20 mM citrate at about pH 6.3, about 263 mM sucrose, about 2 mM calcium chloride, and about 0.05% polysorbate 20. In another aspect, the disclosure features a reconstituted solution made by any of the methods for generating a pharmaceutical solution described herein.

[0088] In yet another aspect, the disclosure features a method for preventing or reducing the progression of vascular calcification in a subject in need thereof, the method including administering to the subject any of the reconstituted formulations described herein in an amount effective to prevent the progression of vascular calcification or reduce vascular calcification in the subject.

[0089] In another aspect, the disclosure features methods for preventing or reducing the progression of pathological calcification in a subject having an ENPP1 deficiency. In another aspect, the disclosure features methods for treating a subject having an ENPP1 deficiency. These methods include administering to the subject any of the reconstituted formulations described herein in an amount effective to prevent the progression of pathological calcification or reduce pathological calcification in the subject.

[0090] In another aspect, the disclosure features a method for preventing or reducing the progression of pathological calcification in a subject having ABCC6 deficiency. In another aspect, the disclosure features a method for treating a subject having ABCC6 deficiency, such as PXE. These methods include administering to the subject any of the reconstituted formulations described herein in an amount effective to prevent the progression of pathological calcification or reduce pathological calcification in the subject.

[0091] In some embodiments, the reconstituted formulation may be administered to a subject at a dose of about 0.1 mg per kilogram of subject, about 0.2 mg per kilogram of subject, about 0.6 mg per kilogram of subject, or about 1.8 mg per kilogram of subject.

[0092] In some embodiments, the reconstituted formulation may be administered to a subject at a dose of about 0.1 mg per kilogram, about 0.2 mg per kilogram, about 0.3 mg per kg, about 0.4 mg per kg, about 0.5 mg per kg, about 0.6 mg per kg, about 0.7 mg per kg, about 0.8 mg per kg, about 0.9 mg per kg, about 1 mg per kg, about 1.1 mg per kg, about 1.2 mg per kg, about 1.3 mg per kg, about 1.4 mg per kg, about 1.5 mg per kg, about 1.6 mg per kg, about 1.7 mg per kg, or 1.8 mg / kg of the subject.

[0093] In yet another aspect, the disclosure features a method for preventing the progression of or reducing tissue calcification in a subject in need thereof, comprising administering to the subject any of the reconstituted formulations described herein in an amount effective to prevent the progression of or reduce tissue calcification in the subject. In some embodiments, the subject has or is suspected of having an ENPP1 deficiency. In some embodiments, the subject has or is suspected of having an ABCC6 deficiency (e.g., pseudoxanthoma elasticum (PXE)).

[0094] In yet another aspect, the disclosure features a method for preventing or reducing the progression of pathological ossification in a subject in need thereof. The method includes administering to the subject any of the reconstituted formulations described herein in an amount effective to prevent or reduce the progression of tissue calcification in the subject. In some embodiments, the subject has or is suspected of having ENPP1 deficiency. In some embodiments, the subject has or is suspected of having ABCC6 deficiency (e.g., pseudoxanthoma elasticum (PXE)).

[0095] In yet another aspect, the disclosure features a method for increasing circulating pyrophosphate (PPi) in a subject in need thereof. The method includes administering to the subject any of the reconstituted formulations described herein in an amount effective to increase circulating PPi in the subject. In some embodiments, the subject has or is suspected of having ENPP1 deficiency. In some embodiments, the subject has or is suspected of having ABCC6 deficiency (e.g., pseudoxanthoma elasticum (PXE)). The phrase "NPP1 deficient" or "ENPP1 deficient" refers to a reduction in the amount of NPP1 protein or NPP1 activity relative to normal serum levels of NPP1 protein or normal activity of NPP1, such reduction resulting in a disease or disorder of pathological calcification and / or pathological ossification and / or a reduction in PPi levels. Such pathological diseases include, but are not limited to, GACI and ARHR2. ENPP1 deficiency, as used herein, does not refer to a slight reduction in the amount of NPP1 protein and / or NPP1 activity that does not result in a disease or disorder of pathological calcification and / or pathological ossification. The phrase "ABCC6 deficient patient" or "ABCC6 deficient subject", as used herein, refers to a patient having at least one pathogenic mutation in the ABCC6 gene that affects the activity and / or expression of the ABC66 protein.

[0096] In another aspect, the disclosure features a method for increasing pyrophosphatase activity in a subject in need thereof. The method includes administering to the subject any of the reconstituted formulations described herein in an amount effective to increase circulating PPi in the subject. In some embodiments, the subject has or is suspected to have ENPP1 deficiency. In some embodiments, the subject has or is suspected to have ABCC6 deficiency (e.g., pseudoxanthoma elasticum (PXE)). The term "pyrophosphatase activity" refers to the ability of the ENPP1 enzyme to hydrolyze the pyrophosphate bond between two phosphate groups in a molecule such as ATP.

[0097] In some embodiments of any of the methods described herein, the subject has a disorder associated with pathologic calcification. In some embodiments of any of the methods described herein, the subject has a disorder associated with pathologic ossification. In some embodiments of any of the methods described herein, the subject has or is at risk of developing pathologic soft tissue calcification, arterial calcification, vascular calcification, chronic kidney disease (CKD), end-stage renal disease (ESRD), uremic arteriolar calcification (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), or hypophosphatemic rickets.

[0098] In yet another aspect, the disclosure features a method for treating or ameliorating one or more symptoms of ENPP1 deficiency in a subject, the method includes administering to the subject any of the reconstituted formulations described herein to ameliorate one or more symptoms of ENPP1 deficiency in the subject.

[0099] In another aspect, the disclosure features a method for treating a subject having an ENPP1 deficiency, the method including administering to the subject any of the reconstituted formulations described herein, thereby treating the subject.

[0100] In yet another aspect, the disclosure features a method for treating or ameliorating one or more symptoms of ABCC6 deficiency in a subject, the method includes administering to the subject any of the reconstituted formulations described herein in an amount effective to ameliorate one or more symptoms of ABCC6 deficiency in the subject.

[0101] In another aspect, the disclosure features a method for treating a subject having an ABCC6 deficiency, the method including administering to the subject a reconstituted formulation in an amount effective to treat the subject.

[0102] In some embodiments of any of the methods described herein, the reconstituted formulation is administered parenterally. In some embodiments of any of the methods described herein, the reconstituted formulation is administered via subcutaneous injection. In some embodiments of any of the methods described herein, the reconstituted formulation is administered via intravenous injection. In some embodiments of any of the methods described herein, the reconstituted formulation is administered via intradermal injection. In some embodiments of any of the methods described herein, the reconstituted formulation is administered via intramuscular injection.

[0103] In some embodiments of any of the methods described herein, the reconstituted formulation is self-administered. In some embodiments of any of the methods described herein, the formulation is administered several times a day, every other day, every third day, weekly, or monthly.

[0104] In some embodiments of any of the methods described herein, a second dose (or additional dose) of the formulation is administered after a suitable time interval (from the first or previous dose) of at least 2 days, 4 days, 1 week, or 1 month. [Brief description of the drawings]

[0105] [Figure 1] The complete unprocessed amino acid sequence of the wild-type ENPP1 precursor protein (SEQ ID NO:1) is shown. The cytoplasmic and transmembrane regions are underlined. Potential N-glycosylation sites are in bold. PSCAKE (residues 99-104, boxed) marks the beginning of the soluble ENPP1 protein portion that includes SMB1 (residues 104-144) and SMB2 (residues 145-189). [Diagram 2] The cytoplasmic domain (residues 1-77), transmembrane domain (TM) (residues 77-100), somatomedin B-like domain 1 (SMB1) (residues 100-142), somatomedin B-like domain 2 (SMB2) (residues 142-187), catalytic domain, and nuclease domain of human ENPP1 are shown. [Diagram 3] The amino acid sequence of the soluble ENPP1 polypeptide (SEQ ID NO:2) is shown. [Figure 4] 4A and 4B show a multiple alignment of various vertebrate soluble ENPP1 polypeptides and human soluble ENPP1 polypeptides (SEQ ID NOs: 4-8). The various soluble ENPP1 polypeptides correspond to the following species and represent regions of specific NCBI accession numbers: mouse (NCBI accession NP_001295256.1, SEQ ID NO: 4), bovine (NCBI accession NP_001193141, SEQ ID NO: 5), rabbit (NCBI accession NP_001162404.1, SEQ ID NO: 6), human (NCBI accession NP_006199.2, SEQ ID NO: 1), and baboon (NCBI accession NP_001076211.2, SEQ ID NO: 8). [Diagram 5] 1 is a bar graph showing the results of size exclusion chromatography (SEC) of ENPP1-Fc formulations containing various buffer components. Data is presented as % high molecular weight species (HMW) or % main peak. [Figure 6] FIG. 1 is a bar graph showing percent recovery for reconstitution of lyophilized ENPP1-Fc polypeptide formulations containing various combinations of buffer and excipient components. [Figure 7] 1 is a bar graph showing a summary of enzyme activity data for lyophilized ENPP1-Fc polypeptide formulations containing various combinations of buffer and excipient components at 5° C. and 40° C. Activity data is expressed in U / mg. [Figure 8] 1 is a bar graph showing a summary of SEC data for lyophilized ENPP1-Fc polypeptide formulations containing various combinations of buffer and excipient components at 5° C. and 40° C. Data is presented as the percentage abundance of high molecular weight species (HMW), major peak species, or low molecular weight species (LMW). [Figure 9]9A and 9B are bar graphs showing a summary of SEC data for lyophilized ENPP1-Fc polypeptide formulations with various buffer, excipient, and surfactant components. The illustrated formulations were tested under stress conditions including agitation at 600 rpm (illustrated as "agitation") or five rounds of freeze-thaw cycles (i.e., storage at -80°C for ≥ 60 min followed by thawing at room temperature; illustrated as "F / T"). Data are presented as HMW% (Figure 9A) or main peak% (Figure 9B). [Figure 10] 1 is a bar graph showing a summary of SEC data for lyophilized ENPP1-Fc polypeptide formulations with various cofactors in a succinate and sucrose buffer / additive background. Data represents the abundance of HMW and major peak species at 5° C. [Figure 11] 1 is a bar graph showing a summary of SEC data for lyophilized ENPP1-Fc polypeptide formulations containing various cofactors in a succinate, sucrose, and PS20 buffer / additive / surfactant background. The data represents the abundance of HMW and major peak species incubated at 40° C. for approximately 6 days. [Figure 12] 12A and 12B are bar graphs showing a summary of SEC data for lyophilized ENPP1-Fc polypeptide formulations containing various cofactors in the context of succinate, sucrose, and PS20 buffer / additive / surfactant. The data represent the abundance of HMW, LMW, and major peak species at 5° C. (FIG. 12B) or incubated at 40° C. for approximately 6 days (FIG. 12A). [Figure 13] 1 is a bar graph showing a summary of osmolality data for lyophilized ENPP1-Fc polypeptide formulation samples. [Figure 14] 1 is a bar graph showing a summary of SEC data for lyophilized ENPP1-Fc polypeptide formulation samples containing several preferred buffer / excipient / additive / surfactant combinations stored for up to 13 weeks at −75° C., 5° C., or 40° C. Data represents the abundance of HMW and major peak species across different formulations / conditions. [Figure 15]1 is a plot showing a summary of SEC data for two preferred lyophilized ENPP1 polypeptide formulations (stable form A and stable form B) stored at either 5° C., 25° C., or 40° C. over a three month period. [Figure 16] 1 is a plot showing a summary of enzyme activity data for two preferred lyophilized ENPP1 polypeptide formulations (stable form A and stable form B) stored at either 5° C., 25° C., or 40° C. over a three month period. [Figure 17] 1 is a plot showing a summary of SEC data for lyophilized ENPP1-Fc polypeptide formulations stored at 5° C. for different periods of time. [Figure 18] 1 is a plot showing a summary of SEC data for lyophilized ENPP1-Fc polypeptide formulations stored at 25° C. for different periods of time. [Figure 19] 1 is a plot showing a summary of SEC data for lyophilized ENPP1-Fc polypeptide formulations stored at 40° C. for different periods of time. [Figure 20] 1 is a plot showing a summary of enzyme activity data for lyophilized ENPP1-Fc polypeptide formulations stored for different periods of time, including at 5° C., 25° C., and 40° C. [Figure 21] 1 is a bar graph showing the recovery rate for reconstituted formulations of lyophilized ENPP1-Fc polypeptide stored in syringes or vials at different time intervals. [Figure 22] 1 is a bar graph showing a summary of enzyme activity data for lyophilized ENPP1-Fc polypeptides stored in syringes or vials at different time intervals. [Diagram 23] 1 is a plot showing the recovery rate for reconstituted formulations of lyophilized ENPP1-Fc polypeptide stored in syringes or vials for different time intervals. [Figure 24] 1 is a plot showing a summary of enzyme activity data for lyophilized ENPP1-Fc polypeptide stored in syringes or vials at different time intervals. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0106] 1. Overview The present application provides stable lyophilized formulations comprising ENPP1 polypeptides and their use in treating diseases associated with ENPP1. In certain aspects, the present disclosure relates to lyophilized formulations of ENPP1 polypeptides and their use (e.g., treating, preventing, or reducing the progression rate and / or severity of pathological calcification and / or ossification, or one or more complications of pathological calcification and / or ossification). The present application provides lyophilized formulations that exhibit enhanced stability, increased shelf life, and lower levels of high molecular weight species (HMW) and aggregates. HMW species may be dimers, tetramers, or higher order aggregates (HMW1 / HMW2).

[0107] ENPP1 polypeptide has been shown to be effective in treating certain diseases of ectopic tissue calcification. ENPP1-Fc has been shown to reduce systemic arterial calcification in a mouse model of GACI (generalized arterial calcification of infancy), a severe disease that occurs in infants and involves widespread arterial calcification (Albright, et al., 2015, Nature Comm.10006). Fusion proteins of ENPP1 have also been described to treat severe tissue calcification diseases (see, for example, PCT Publication Nos. 2014 / 126965 and 2016 / 187408), and fusion proteins of ENPP1 containing negatively charged bone targeting domains have been described to treat GACI (PCT Publication Nos. 2011 / 113027 and 2012 / 125182).

[0108] Mammalian ENPP1 polypeptides, variants, or variant fragments thereof have been previously disclosed in International PCT Publication Nos. 2014 / 126965 - Braddock et al., 2016 / 187408 - Braddock et al., 2017 / 087936 - Braddock et al., and 2018 / 027024 - Braddock et al., all of which are incorporated by reference in their entireties herein.

[0109] 2. Lyophilized formulations In certain aspects, the present disclosure contemplates the use of lyophilized ENPP1 formulations for the treatment or prevention of a disease or condition associated with abnormal activity of an ENPP1 polypeptide.

[0110] In one aspect, the ENPP1 formulation of the present disclosure is lyophilized. During lyophilization, the ENPP1 polypeptide is converted from an aqueous phase to an amorphous solid phase, which is believed to protect the protein from chemical and / or conformational instability. Lyophilization is performed using techniques common in the art, and the lyophilized formulation is optimized for stability, shelf life, and reduced levels of high molecular weight (HMW) species and aggregates. Tang et al., Pharm Res. 21:191-200, (2004) and Chang et al., Pharm Res. 13:243-9 (1996). The disclosed lyophilized ENPP1 formulation helps stabilize the protein against the stresses of manufacturing, shipping, and storage. The excipients and additives used in the lyophilized formulation are essential components of the formulation and therefore need to be safe and well tolerated by patients. For protein drugs, the choice of excipients and additives is particularly important as it can affect both the efficacy and immunogenicity of the drug. Excipients and additives are also useful for reducing the viscosity of high concentration ENPP1 polypeptide formulations to allow their delivery and enhance patient convenience. The formulations, excipients, and additives disclosed herein provide stability against these stresses. Common excipients are known in the art and can be found in Powell et al., Compendium of Excipients fir Parenteral Formulations (1998), PDA J.Pharm.Sci.Technology, 52:238-311.

[0111] In certain embodiments, the ENPP1 formulation comprises a stabilizer. These stabilizers can be classified based on their mechanism of stabilizing proteins against various chemical and physical stresses. Some stabilizers are used to reduce the effects of certain stresses or to regulate the specific sensitivity of certain proteins. Other stabilizers have a more general effect on the physical and covalent stability of proteins. Given the teachings and guidance provided herein, one of ordinary skill in the art will understand how much or what range of stabilizer can be included in any particular formulation to achieve an ENPP1 formulation of the present disclosure that is likely to promote the retention and stability of the ENPP1 polypeptide.

[0112] In certain embodiments, the ENPP1 formulations disclosed herein include bulking agents. Such fillers are included for the purposes of long-term stabilization, bulking up solid formulations containing small amounts of potent active ingredients (hence often referred to as "bulking agents," "fillers," or "diluents"), or imparting therapeutic enhancements to the active ingredients in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility.

[0113] The ENPP1 formulations disclosed herein include buffers, stabilizers, surfactants, sugars, salts, and amino acids, which are described in more detail below.

[0114] Those skilled in the art will recognize that the concentrations of excipients described herein share interdependencies within a particular formulation. As an example, the concentration of bulking agent is reduced in one embodiment, for example, when the protein concentration is high, or, for example, when the stabilizer concentration is high. In addition, those skilled in the art will recognize that the concentration of stabilizer can be increased as appropriate (i.e., a "tonicity" amount of stabilizer can be used) to maintain the isotonicity of a particular formulation without bulking agent. Excipients and other additives are added to impart or enhance manufacturability and / or final product quality, such as stability and delivery of drug products (e.g., proteins). The ENPP1 formulations disclosed herein include suitable excipients that enhance suitable stability, safety, and marketability.

[0115] In certain embodiments, the lyophilized ENPP1 formulation comprises at least one or more of a buffer, bulking agent, stabilizer, and / or surfactant. In some embodiments, the surfactant is selected to help reduce the formation of HMW species during the lyophilization step or when aggregation during reconstitution is an issue. A suitable buffer is included to keep the formulation within a stable pH zone during manufacturing (e.g., dilution, sterile filtration, filling, etc.) and after reconstitution of the lyophilized product. The following table provides certain excipient components useful for lyophilized protein formulations. [Table 1]

[0116] (a) Buffers and buffering agents Typically, the stability of pharmacologically active polypeptide formulations is observed to be maximum in a narrow pH range. This pH range of optimal stability needs to be identified early during pre-formulation testing. Several approaches, such as accelerated stability testing and calorimetric screening testing, are useful in this endeavor (Remmele RL Jr., et al., Biochemistry, 38(16):5241-7(1999)). Once the formulation is complete, the protein must be manufactured and maintained throughout its shelf life. Therefore, buffers are almost always used to control the pH in the formulation.

[0117] Several factors must be considered when selecting a buffer. First, the buffer type and its concentration must be defined based on its pKa and the desired formulation pH. Equally important is that the buffer is compatible with proteins and other formulation excipients and does not catalyze degradation reactions. A third important aspect to consider is the stinging and irritation that the buffer may induce upon administration. For drugs administered via subcutaneous (SC) or intramuscular (IM) routes, the possibility of stinging and irritation is high, where the drug solution remains at the site for a relatively longer period than when administered via IV routes, where the formulation is rapidly diluted in the blood upon administration. For formulations administered by direct IV infusion, the total amount of buffer (and any other formulation components) must be monitored. Particular attention must be paid to potassium ions administered in the form of potassium phosphate buffer, which may induce cardiovascular effects in patients (Hollander-Rodriguez JC, et al., Am. Fam. Physician., 73(2):283-90 (2006)).

[0118] Buffers for lyophilized formulations require further consideration. Some buffers, such as sodium phosphate, can crystallize from the protein amorphous phase during freezing, resulting in a shift in pH. Other common buffers, such as acetate and imidazole, can sublime or evaporate during the lyophilization process, thereby shifting the pH of the formulation during lyophilization or after reconstitution.

[0119] In certain embodiments, exemplary buffering agents used to buffer the ENPP1 formulations described herein include, but are not limited to, organic acids, succinates, phosphates, acetates, citrates, Tris, HEPES, and amino acids or mixtures of amino acids, including, but not limited to, aspartic acid, histidine, arginine, and glycine. In certain embodiments, the buffering agents are succinates, citrates, and phosphates. In one embodiment, the buffer system present in the ENPP1 formulation is physiologically compatible and selected to maintain the desired pH of the pharmaceutical formulation. In another embodiment, the pH of the solution is between pH 2.0 and pH 12.0. For example, in various embodiments, the pH of the solution may be 5.5, 5.7, 6.0, 6.3, 6.5, 6.7, 7.0, 7.3, 7.5, 7.7, 8.0, 8.3, 8.5, 8.7, 9.0, 9.3, 9.5, 9.7, or 10.0.

[0120] The pH buffering compound may be present in any amount suitable to maintain the pH of the ENPP1 formulation at a predetermined level. When an appropriately low level of buffer is used, crystallization and pH shifts may be avoided. In one embodiment, the pH buffer concentration is 0.1 mM to 500 mM (1 M). For example, it is contemplated that 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, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or 500 mM. In some embodiments, the buffering agent maintains a pH range of pH 6-7 when reconstituted in solution. In some embodiments, the buffering agent maintains a pH range of pH 7-8 when reconstituted in solution.

[0121] Exemplary pH buffers used to buffer the ENPP1 formulations described herein include, but are not limited to, organic acids, succinates, phosphates, acetates, citrates, Tris, HEPES, and amino acids or mixtures of amino acids. In some embodiments, the buffer reduces the formation of high molecular weight species by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%.

[0122] (b) Stabilizers and bulking agents In one embodiment of the pharmaceutical formulation, a stabilizer (or combination of stabilizers) is added to prevent or reduce storage-induced aggregation and chemical degradation. A hazy or turbid solution upon reconstitution usually indicates that the protein has precipitated or at least aggregated. The term "stabilizer" refers to an excipient that can prevent aggregation or chemical degradation (e.g., autolysis, deamidation, oxidation, etc.). In certain embodiments, the ENPP1 formulations provided herein include stabilizers such as, but not limited to, sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, polyhydroxy compounds (including polysaccharides such as dextran, starch, hydroxyethyl starch, cyclodextrin, N-methylpyrrolidone, cellulose, and hyaluronic acid) [Carpenter et al., Develop.Biol.Standard 74:225, (1991)]. In one embodiment of the disclosure, sucrose and mannitol are used as stabilizers in the ENPP1 formulations disclosed herein. In another embodiment of the disclosure, sucrose is used as a stabilizer.

[0123] In certain embodiments, the ENPP1 formulation comprises a stabilizer at a concentration of about 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, 900, or 1000 mM. Similarly, in certain embodiments of the present disclosure, the stabilizer is incorporated at a concentration of about 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 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, or 20% w / v. In other embodiments, the ENPP1 formulation also includes an appropriate amount of a bulking agent and an osmolality adjusting agent. In various embodiments of the present disclosure, the bulking agent includes a polymer such as, for example, dextran, polyvinylpyrrolidone, carboxymethylcellulose, lactose, sorbitol, trehalose, or xylitol. In various embodiments of the present disclosure, the bulking agent is incorporated at a concentration of about 0.1, 0.5, 0.7, 0.8, 0.9, 1.0, 1.2, 1.5, 1.7, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 700, 900, or 1000 mM.

[0124] (c) Surfactants In certain embodiments, the ENPP1 formulations disclosed herein may additionally comprise a surfactant. Surfactants are commonly used in protein formulations to prevent surface-induced degradation. Surfactants are amphiphilic molecules that have the ability to win the competition with proteins for interfacial positions (and / or promote proper refolding of structurally modified protein molecules). The hydrophobic portion of the surfactant molecule occupies the interfacial positions (e.g., gas / liquid), while the hydrophilic portion of the molecule remains oriented toward the bulk solvent. At sufficient concentrations (typically around the detergent's critical micelle concentration), the surface layer of the surfactant molecule serves to prevent protein molecules from adsorbing at the interface, thereby minimizing surface-induced degradation. Surfactants contemplated herein include, but are not limited to, fatty acid esters of sorbitan polyethoxylate, i.e., polysorbate 20 and polysorbate 80. The two differ only in the length of the aliphatic chain that confers hydrophobicity to the molecules C-12 and C-18, respectively. Thus, polysorbate 80 has a higher surface activity and a lower critical micelle concentration than polysorbate 20.

[0125] Detergents can also affect the thermodynamic conformational stability of proteins. Non-ionic detergents are generally useful for protein stabilization. Ionic detergents (detergents) usually destabilize proteins. Again, the effect of a given detergent excipient will be protein specific. For example, polysorbates have been shown to decrease the stability of some proteins and increase the stability of others. Detergent destabilization of proteins can be theoretically explained in terms of the hydrophobic tails of the detergent molecules that may be involved in specific binding with partially or fully unfolded protein states. These types of interactions may cause a shift in the conformational equilibrium towards more extended protein states (i.e., increasing the exposure of hydrophobic portions of the protein molecule, complementing the binding to polysorbates). Alternatively, if the protein native state exhibits some hydrophobic surface, detergent binding to the native state may stabilize that conformation. Another aspect of polysorbates is that they are inherently susceptible to oxidative degradation. Often, as raw materials, they contain sufficient amounts of peroxides to cause oxidation of protein residue side chains, especially methionine. The potential for oxidative damage from the addition of stabilizers emphasizes that the lowest effective concentration of an excipient should be used in the formulation. For surfactants, the effective concentration for a given protein depends on the mechanism of stabilization.

[0126] Surfactants are also added in an appropriate amount to prevent surface-related aggregation phenomena during freezing and drying [Chang, B, J. Pharm. Sci. 85:1325, (1996)]. Thus, exemplary surfactants include anionic, cationic, nonionic, zwitterionic, and amphoteric surfactants, including, but not limited to, surfactants derived from natural amino acids. Anionic surfactants include, but are not limited to, sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate, chenodeoxycholic acid, N-lauroyl sarcosine sodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate, and sodium glycodeoxycholic acid salt. 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. Surfactants include, but are not limited to, 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 dioleylphosphatidylcholine (DOPC), dimyristoylphosphatidylglycerol (DMPG), dimyristoylphosphatidylcholine (DMPC), and (dioleylphosphatidylglycerol) DOPG; sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Thus, compositions are further provided that include these surfactants individually or as a mixture in different ratios. In one embodiment of the present disclosure, the surfactant is TWEEN-80. In the present formulation, the surfactant is incorporated at a concentration of about 0.01 to about 0.5 g / L.In the formulations provided herein, in various embodiments, the surfactant concentration is 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 g / L. Similarly, in certain embodiments of the present disclosure, the surfactant is incorporated at a concentration of about 0.001, 0.002, 0.003, 0.004, 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.5, 0.7, 0.8, 0.9, or 1.0% w / v.

[0127] (d) Salt In certain embodiments, the ENPP1 formulations disclosed herein include salts. Salts are often added to increase the ionic strength of the formulation, which can be important for protein solubility, physical stability, and isotonicity. Salts can affect the physical stability of proteins in a variety of ways. Ions can stabilize the native state of a protein by binding to charged residues on the surface of the protein. Alternatively, salts can stabilize the denatured state by binding to peptide groups along the protein backbone (--CONH--). Salts can also stabilize the protein native structure by shielding repulsive electrostatic interactions between residues within the protein molecule. Salts in protein formulations can also shield attractive electrostatic interactions between protein molecules that can lead to protein aggregation and insolubility. Salts (i.e., electrolytes) can make it more difficult to lyophilize the formulation. For this reason, only enough salt should be included in the formulation to maintain protein structure stability, and typically the level of electrolytes is very low.

[0128] In certain embodiments, ENPP1 formulations disclosed herein may include salts, such as, for example, sodium chloride (NaCl), calcium chloride (CaCl2), zinc chloride (ZnCl2), and / or magnesium chloride (MgCl2) salts. In certain embodiments, ENPP1 formulations disclosed herein have a salt concentration of between 0.0 (i.e., no salt), 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.010, 0.011, 0.012, 0.013, 0.014, 0.015, 0.020, 0.050, 0.080, 0.1, 1, 10, 20, 30, 40, 50, 80, 100, 120, 150, 200, 300, and 500 mM. In one embodiment of the present disclosure, 0.0 mM NaCl (ie, no NaCl) is included in the formulation.

[0129] (e) Amino acid Amino acids have been found to be versatile in protein formulations as buffers, bulking agents, stabilizers, and antioxidants. Thus, in one aspect, the ENPP1 formulations disclosed herein include amino acids such as, for example, glycine, arginine, histidine, alanine, proline, serine, and glutamic acid. These amino acids often provide multiple benefits to polypeptide formulations. Histidine is commonly found in commercial protein formulations, and this amino acid provides an alternative to citrate, a buffer known to cause a stinging sensation upon injection. Interestingly, histidine has also been reported to have a stabilizing effect on aggregation when used at high concentrations in both liquid and lyophilized forms (Chen B, et al., Pharm Res., 20(12):1952-60(2003)). Histidine has also been observed by others to reduce the viscosity of high protein concentration formulations. In another aspect, a formulation is provided that includes one or more of the amino acids glycine, arginine, and alanine, which have been shown to stabilize proteins by a mechanism of preferential exclusion. Glycine is also a commonly used bulking agent in lyophilized formulations. Arginine has been shown to be an effective agent for inhibiting aggregation and has been used in both liquid and lyophilized formulations. In the ENPP1 formulations provided, the amino acid concentrations are 0.1, 1, 10, 20, 30, 40, 50, 80, 100, 120, 150, 200, 300, and 500 mM. In one embodiment of the present disclosure, the amino acid is glycine.

[0130] (f) Antioxidants Oxidation of protein residues occurs from a number of different sources. In addition to adding specific antioxidants, prevention of oxidative protein damage involves careful control of a number of factors throughout the manufacturing process and throughout storage of the product, such as atmospheric oxygen, temperature, light exposure, and chemical contamination. Thus, the present disclosure contemplates the use of pharmaceutical antioxidants, including but not limited to reducing agents, oxygen / free radical scavengers, or chelating agents. Antioxidants in therapeutic protein formulations, in one aspect, are water-soluble and remain active throughout the product shelf life. Reducing agents and oxygen / free radical scavengers function by scavenging reactive oxygen species in solution. In various embodiments of the formulations provided herein, the antioxidant concentration is 0.005, 0.01, 0.02, 0.03, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 mg / mL.

[0131] (g) Metal ions Generally, transition metal ions are undesirable in protein formulations because they can catalyze physical and chemical decomposition reactions in proteins. However, certain metal ions are included in formulations where they are cofactors of proteins, and in suspension formulations of proteins where they form coordination complexes (e.g., insulin in zinc suspensions). Recently, it has been proposed that the use of magnesium ions (10-120 mM) inhibits the isomerization of aspartic acid to isoaspartic acid (WO2004 / 039337).

[0132] (h) Preservatives Preservatives are necessary when developing multi-use parenteral formulations with more than one extraction from the same container. Their main function is to inhibit microbial growth and ensure sterility of the product throughout the shelf life or use of the drug product. Commonly used preservatives include, but are not limited to, benzyl alcohol, phenol, and m-cresol. Although preservatives have a long history of use, developing protein formulations containing preservatives can be difficult. Preservatives almost always have a destabilizing effect on proteins (aggregation), which is a major factor limiting their use in multi-dose protein formulations (Roy S, et al., J Pharm Sci., 94(2):382-96 (2005)). When practical, preservatives should be included in the diluent formulation and not in formulations that are lyophilized.

[0133] In certain embodiments, the soluble ENPP1 polypeptide is formulated as a lyophilized polypeptide formulation comprising a therapeutic amount of the soluble ENPP1 polypeptide disclosed herein, whereby the lyophilized polypeptide formulation can be reconstituted into a liquid form solution. In some embodiments, the lyophilized polypeptide formulation is reconstituted in a sterile injection solution. In some embodiments, the lyophilized polypeptide formulation is reconstituted in a reconstitution solution. In some embodiments, the reconstitution solution comprises a pharma- ceutically acceptable carrier and / or additive. In some embodiments, the pharma-ceutically acceptable carrier is selected from saline, purified water, or sterile water for injection. In some embodiments, the formulation is completely reconstituted within a period of less than 100 seconds, 80 seconds, 70 seconds, 69 seconds, 68 seconds, 67 seconds, 66 seconds, 65 seconds, 64 seconds, 63 seconds, 62 seconds, 61 seconds, or 60 seconds.

[0134] In certain embodiments, the lyophilized polypeptide formulation comprises an ENPP1 polypeptide. In some embodiments, the lyophilized polypeptide comprises a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the lyophilized polypeptide comprises a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence at least 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a portion of a polypeptide having the amino acid sequence of SEQ ID NO: 2. In some embodiments, the ENPP1 polypeptide is a fusion protein. In some embodiments, the ENPP1 polypeptide is a fusion protein further comprising an Fc domain of an immunoglobulin. In some embodiments, the Fc domain of the immunoglobulin is an Fc domain of an IgG1 immunoglobulin. In some embodiments, the ENPP1 polypeptide is a fusion protein comprising a soluble ENPP1 polypeptide domain and one or more heterologous protein moieties. In some embodiments, the heterologous protein moieties increase the circulating half-life of the soluble ENPP1 polypeptide in a mammal. In some embodiments, the heterologous protein moieties comprise an Fc domain. In some embodiments, the Fc domain comprises a polypeptide comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the ENPP1 polypeptide further comprises a heterologous moiety. In some embodiments, the heterologous moiety is selected from the group consisting of a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, and a lipid moiety.

[0135] In certain embodiments, the lyophilized polypeptide formulation comprises a buffering agent. In certain embodiments, the buffering agent maintains the pH in the range of pH 5.0 to pH 8.0, in the range of about pH 5.5 to pH 7.5, in the range of about pH 6.0 to pH 7.0, in the range of about pH 6.0 to pH 6.5. In some embodiments, the buffering agent maintains a pH of 6.5±0.5. In a preferred embodiment, the pH is pH 6.3. In certain embodiments, the buffering agent is selected from the group consisting of succinate, citrate, bicarbonate, phosphate, Tris, or glycylglycine. In certain embodiments, the buffering agent is succinate, citrate, or phosphate. In a preferred embodiment, the buffering agent is citrate. The buffering agent may range in concentration from 5 mM to 100 mM when the lyophilized polypeptide formulation is reconstituted in solution. In certain embodiments, the buffering agent is present at a concentration in the range of 10 mM to 50 mM, or 15 mM to 25 mM. In certain preferred embodiments, the buffering agent is present at a concentration of 20 mM. The buffering agent may confer increased stability of the ENPP1 polypeptide in both lyophilized and reconstituted forms. In certain embodiments, the buffering agent may be selected to facilitate increasing the onset temperature of aggregate formation. In some embodiments, the buffering agent increases the onset temperature of aggregate formation by at least 1° C., 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., or 10° C. In certain embodiments, the buffering agent may reduce the formation of high molecular weight species. In some embodiments, the buffering agent reduces the formation of high molecular weight species by at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the buffering agent reduces the formation of high molecular weight species by at least 10%-100%, 20%-80%, 30%-70%, or 40%-60%. In some embodiments, the buffer reduces the formation of high molecular weight species by at least 50%.

[0136] The lyophilized polypeptide formulations of the present disclosure may include one or more pharma- ceutically acceptable additives and / or stabilizers. In certain embodiments, the pharma-ceutically acceptable additives include amino acids, salts, sugars, and / or polyols. In some embodiments, the pharma-ceutically acceptable additives are arginine, proline, and / or glycine. In preferred embodiments, the pharma-ceutically acceptable additives are stabilizers such as sucrose and / or mannitol. In certain embodiments, the one or more pharma-ceutically acceptable additives, when reconstituted in solution, have a concentration ranging from 50 mM to 300 mM. In some embodiments, the one or more pharma-ceutically acceptable additives, when reconstituted in solution, have a concentration ranging from 50 mM to 100 mM, 60 mM to 90 mM, or 75 mM to 85 mM. In some embodiments, the one or more pharma- ceutically acceptable excipients, when reconstituted in solution, have a concentration of 50 mM, 60 mM, 70 mM, 80 mM, 82 mM, 84 mM, 86 mM, 88 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 171 mM, 172 mM, 173 mM, 174 mM, 175 mM, 176 mM, 177 mM, 178 mM, 179 mM, 180 mM, 181 mM, 182 mM, 183 mM, 184 mM, 185 mM, 186 mM, 187 mM, 188 mM, 189 mM, 190 mM, 191 mM, 192 mM, 193 mM, 194 mM, 195 mM, 196 mM, 197 mM, 198 mM, 199 mM, 200 mM, 201 mM, 202 mM, 203 mM, 204 mM, 205 mM, 206 mM, 207 mM, 208 mM, 209 mM, 210 mM, 211 mM, 212 mM, 213 mM, 214 mM, 215 mM, 216 mM, 217 mM, 218 mM, 219 mM, 220 mM, 221 mM, 222 mM, 223 mM, 224 mM, 225 mM, 226 mM, 227 mM, 228 mM, 229 mM, 230 mM, 231 mM, 232 mM, 233 mM, 234 mM, , 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 280, 290, 300 mM. The pharma- ceutically acceptable additives and / or stabilizers may confer increased stability of the ENPP1 polypeptide in both lyophilized and reconstituted forms. In certain embodiments, the pharma- ceutically acceptable additive and / or stabilizer reduces the formation of high molecular weight species by at least 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the pharma-ceutically acceptable additive and / or stabilizer reduces the formation of high molecular weight species by at least 10%-100%, 20%-80%, 30%-70%, or 40%-60%.In some embodiments, the pharma- ceutically acceptable excipient and / or stabilizer reduces the formation of high molecular weight species by at least 50%.

[0137] The ENPP1 polypeptide of the present disclosure comprises a cofactor coordinated within the ENPP1 nuclease-like domain and the SMB-like domain (see, e.g., Kato K. et al., Proc Natl Acad Sci USA. 2012; 109(42): 16876-81). In certain embodiments, the lyophilized polypeptide formulation of the present disclosure may comprise one or more ENPP1 polypeptide cofactors. In some embodiments, the ENPP1 polypeptide cofactor comprises calcium, zinc, and / or adenosine monophosphate. In some embodiments, the ENPP1 polypeptide cofactor is CaCl2, CaSO4, ZnCl2, ZnSO4, and / or adenosine monophosphate. In some embodiments, the ENPP1 polypeptide cofactor is CaCl2 and / or adenosine monophosphate. In a preferred embodiment, the cofactor ENPP1 polypeptide cofactor is CaCl2. In some embodiments, the ENPP1 polypeptide cofactor, when reconstituted in solution, has a concentration ranging from 1 mM to 10 mM, 1 mM to 5 mM, or 1 mM to 3 mM. In preferred embodiments, the ENPP1 polypeptide cofactor has a concentration within ±1 mM of 2 mM. In even more preferred embodiments, the ENPP1 polypeptide cofactor has a concentration of 2 mM.

[0138] In some embodiments, the compositions and formulations include a surfactant. In some embodiments, the surfactant is a polysorbate, a poloxamer, a triton, sodium dodecyl sulfate, sodium laurel sulfate, sodium octyl glucoside, lauryl sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, lauryl sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroamidopropyl-betaine, cocamide protease, or the like. The surfactants include pyr-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmidopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmidopropyl dimethylamine, isostearamidopropyl-dimethylamine, sodium cocoyl methyl taurate, disodium oleyl methyl taurate, dihydroxypropyl PEG-5 linoleic ammonium chloride, polyethylene glycol, polypropylene glycol, and mixtures thereof. The surfactants can be, for example, but are not limited to, polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, polysorbate 188, PEG3350, and mixtures thereof. In a preferred embodiment, the surfactant is polysorbate 20 (PS20), polysorbate 80 (PS80), or polysorbate 188 (PS188). In a preferred embodiment, the surfactant is polysorbate 20 (PS20).

[0139] The concentration of the surfactant may be expressed as a percentage (w / v). For lyophilized formulations, the concentration % (w / v) represents the surfactant concentration when reconstituted in solution. The surfactant concentration generally ranges from about 0.001% (w / v) to 1% (w / v). In some embodiments, the surfactant concentration ranges from 0.01% to 0.5% (w / v), 0.015% to 0.25% (w / v), 0.02% to 0.1% (w / v). In preferred embodiments, the surfactant concentration ranges from 0.02% to 0.1% (w / v).

[0140] The lyophilized polypeptide formulations of the present disclosure may exhibit increased stability over time. In certain embodiments, the lyophilized polypeptide formulations of the present disclosure exhibit long-term stability at -80°C to 40°C. In some embodiments, the formulations have a shelf life of at least 3, 6, 12, 24, 36, 48, or 60 months. In some embodiments, the reconstituted formulations have a shelf life of at least 1, 2, 3, 4, 5, 6, 12, 18, 24, 48, or 60 hours.

[0141] In some embodiments, the reconstituted formulation is stable at room temperature for at least 8 hours at a concentration ranging from 1 mg / ml to 50 mg / ml, 2 mg / ml to 45 mg / ml, 5 mg / ml to 40 mg / ml, 10 to 30 mg / ml, 15 to 20 mg / ml. In some embodiments, the reconstituted formulation is stable at 5° C. for at least 24 hours at a concentration ranging from 1 mg / ml to 50 mg / ml, 2 mg / ml to 45 mg / ml, 5 mg / ml to 40 mg / ml, 10 to 30 mg / ml, 15 to 20 mg / ml.

[0142] Lyophilized polypeptide formulations disclosed herein may be administered in a reconstituted form. Routes of administration for the reconstituted lyophilized polypeptide formulations disclosed herein include inhalation, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, ​​vaginal (e.g., vaginal and perivaginal), intranasal, (trans)rectal, intravesical, intrapulmonary, intraduodenal, intragastric, intrathecal, subcutaneous, intramuscular, intradermal, intraarterial, intravenous, intrabronchial, inhalation (e.g., aerosol), ophthalmic, respiratory, and topical administration. In a preferred embodiment, the reconstituted formulation is administered parenterally. In some embodiments, the reconstituted formulation is administered parenterally. In some embodiments, the reconstituted formulation is administered via subcutaneous, intravenous, intradermal, or intramuscular injection. In some embodiments, the reconstituted formulation is self-administered. In some embodiments, the lyophilized formulation comprises a therapeutically effective dose. In some embodiments, the formulation is administered several times a day, every 2 days, every 3 days, every week, or every month. In some embodiments, a second dose of the formulation is administered after a suitable time interval of at least 2 days, 4 days, 1 week, or 1 month.

[0143] In certain embodiments, the disclosure provides a kit comprising a lyophilized polypeptide formulation. In some embodiments, the kit comprises a lyophilized polypeptide that may be reconstituted in a sterile injection solution (or sterile water) prior to use. In some embodiments, the kit comprises one or more vials. In some embodiments, the kit comprises one or more vials comprising a lyophilized polypeptide formulation. In some embodiments, the kit comprises an injection device comprising a vial, a reconstitution solution, a syringe, and / or a pre-filled syringe. In some embodiments, the lyophilized polypeptide is reconstituted in a sterile injection solution by transferring the reconstitution solution to the vial. In some embodiments, the lyophilized polypeptide is reconstituted in a sterile injection solution. In some embodiments, the lyophilized polypeptide is reconstituted in a sterile injection solution (or sterile water) prior to use. In some embodiments, the injection device is used to parenterally administer the sterile injection solution. In some embodiments, the sterile injection solution is administered via subcutaneous injection. In some embodiments, the sterile injection solution is administered via intradermal injection. In some embodiments, the sterile injection solution is administered via intramuscular injection. In some embodiments, the sterile injection solution is administered via intramuscular injection. In some embodiments, the sterile injectable solution is self-administered, hi some embodiments, the sterile injectable solution comprises a therapeutically effective dose.

[0144] The lyophilized polypeptide formulations disclosed herein may be used in methods of treating, reversing, or preventing the progression of diseases associated with ENPP1 deficiency disclosed herein. In some embodiments, the formulations are for use in methods of treating, reversing, or preventing the progression of ossification of the posterior longitudinal ligament (OPLL) in a subject in need thereof. In some embodiments, the formulations are for use in methods of treating, reversing, or preventing the progression of hypophosphatemic rickets in a subject in need thereof. In some embodiments, the formulations are for use in methods of treating, reversing, or preventing the progression of ABCC6 deficiency (such as, for example, pseudoxanthoma elasticum (PXE)) in a subject in need thereof. In some embodiments, the formulations are for use in methods of reducing or preventing the progression of age-related arterial sclerosis in a subject in need thereof. In some embodiments, the formulations are for use in methods of treating, reversing, or preventing the progression of calcification of atherosclerotic plaques in vascular arteries in a subject in need thereof. In some embodiments, the formulation is for use in a method of treating, reversing, or preventing the progression of osteoarthritis in a subject in need thereof. In some embodiments, the formulation is for use in a method of treating, reversing, or preventing the progression of arterial sclerosis due to progeria in a subject in need thereof. In some embodiments, the formulation is for use in a method of treating, reversing, or preventing the progression of atherosclerotic plaque calcification in vascular arteries in a subject in need thereof. In some embodiments, the formulation is for use in a method of treating, reversing, or preventing the progression of osteoarthritis in a subject in need thereof. In some embodiments, the formulation is for use in a method of treating, reversing, or preventing the progression of arterial sclerosis due to progeria in a subject in need thereof.In some embodiments, the formulation is for use in a method of treating, reversing, or preventing the progression of X-linked hypophosphatemic rickets (XLH), hereditary hypophosphatemic rickets (HHRH), hypophosphatemic bone disease (HBD), autosomal dominant hypophosphatemic rickets (ADHR), and / or autosomal recessive hypophosphatemic rickets in a subject in need thereof. In some embodiments, the formulation is for use in a method of treating, reversing, or preventing the progression of age-related osteopenia in a subject in need thereof. In some embodiments, the formulation is for use in a method of treating, reversing, or preventing the progression of ankylosing spondylitis in a subject in need thereof. In some embodiments, the formulation is for use in a method of treating, reversing, or preventing the progression of stroke in childhood sickle cell anemia in a subject in need thereof.

[0145] The formulations disclosed herein are lyophilized using techniques well known in the art. Further information regarding lyophilization can be found in Carpenter, JF and Chang, BS, Lyophilization of Protein Pharmaceuticals, Biotechnology and Biopharmaceutical Manufacturing, Processing and Preservation, KEAvis and VLWu, eds. (Buffalo Grove, Interpharm Press, Inc.), pp. 199 264 (1996), U.S. Patent Nos. 7,247,707, 7,087,723, and 6,586,573.

[0146] 3. ENPP1 Polypeptide In certain aspects, the present disclosure relates to lyophilized formulations of ENPP1 polypeptides and their uses. The ENPP1 polypeptides disclosed herein include naturally occurring polypeptides of the ENPP1 family and any variants thereof (including mutants, fragments, fusions, and / or peptidomimetic forms) that retain biological activity. The term "ENPP1" or "ENPP1 polypeptide" refers to ectonucleotide pyrophosphatase / phosphodiesterase 1 protein (NPP1 / ENPP1 / PC-1) and ENPP1-related proteins from any species. ENPP1 proteins include type II transmembrane glycoproteins that form homodimers. Each monomer of the ENPP1 protein contains a short intracellular N-terminal domain involved in targeting to the plasma membrane, a transmembrane domain, and a large extracellular region that contains several domains. The large extracellular region contains the SMB1 and SMB2 domains, which have been reported to be involved in ENPP1 dimerization (R. Gijsbers, H. et al., Biochem. J. 371; 2003: 321-330). Specifically, the SMB domain contains eight cysteine ​​residues, each arranged in four disulfide bonds, and has been shown to mediate ENPP1 homodimerization through covalent intercystine and intramolecular bonds. The protein cleaves a variety of substrates, including phosphodiester bonds of nucleotides and nucleotide sugars, and pyrophosphate bonds of nucleotides and nucleotide sugars. The ENPP1 protein functions to hydrolyze nucleoside 5' triphosphatases to the corresponding monophosphates and also hydrolyze diadenosine polyphosphates. The ENPP1 protein plays a role in purinergic signaling, which is involved in the regulation of cardiovascular, neurological, immunological, musculoskeletal, hormonal, and hematological functions. An exemplary amino acid sequence of human ENPP1 precursor protein (NCBI accession NP_006199) is shown in Figure 1 (SEQ ID NO: 1). Human ENPP1 precursor protein contains an endogenous ENPP1 signal peptide sequence at the ENPP1 N-terminus. Amino acid numbering for all ENPP1-related polypeptides described herein is based on the numbering of the human ENPP1 precursor protein sequence provided in Figure 1, unless otherwise specified.In certain embodiments, the ENPP1 precursor protein further comprises an endogenous or heterologous signal peptide sequence. During proteolysis, the signal peptide sequence is cleaved from the ENPP1 precursor protein to provide the mature ENPP1 protein. See, for example, Jansen S, et al. J Cell Sci. 2005; 118 (Pt 14): 3081-9. Exemplary signal peptide sequences that may be used with the polypeptides disclosed herein include, but are not limited to, the ENPP1 signal peptide sequence, the ENPP2 signal peptide sequence, the azurocidin signal sequence, the ENPP7 signal peptide sequence, and / or the ENPP5 signal peptide sequence. The processed (mature) extracellular ENPP1 polypeptide sequence is shown in Figure 3 (SEQ ID NO: 2).

[0147] It is generally known in the art that ENPP1 is well conserved among vertebrates, and most of the extracellular domain is substantially conserved. For example, Figure 4A and Figure 4B illustrate multiple alignments of human ENPP1 extracellular domain compared to various ENPP1 orthologs. ENPP1, which binds various nucleotide triphosphates (e.g., ATP, UTP, GTP, TTP, and CTP), pNP-TMP, 3',5'-cAMP, and 2'-3'-cGAMP, is also highly conserved (see, for example, Kato K. et al., Proc Natl Acad Sci USA. 2012; 109(42): 16876-81 and Mackenzie NC, et al. Bone. 2012; 51(5): 961-8). Thus, from these alignments, it is possible to predict important amino acid positions with the extracellular domain that are important for normal ENPP1 activity, and to predict amino acid positions that are likely to tolerate substitution without significantly altering normal ENPP1 activity. Thus, a useful active human ENPP1 polypeptide according to the disclosed compositions may contain one or more amino acids at the corresponding positions from the sequence of another vertebrate ENPP1, or may contain similar residues to those of the human or other vertebrate sequence. The substitution of one or more amino acids at the corresponding positions may include conservative modifications or substitutions that are unlikely to alter the shape of the polypeptide chain or alter normal ENPP1 activity. Examples of conservative modifications or substitutions include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine, or methionine, for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamine for aspartic acid, or glutamine for asparagine. For example, ENPP1 polypeptides include polypeptides derived from the sequence of any known ENPP1 polypeptide having a sequence that is at least about 80%, preferably at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the sequence of an ENPP1 polypeptide.

[0148] ENPP1 proteins have been characterized in the art in terms of structural and biological features. In certain embodiments, the soluble ENPP1 proteins disclosed herein comprise pyrophosphatase and / or phosphodiesterase activity. For example, in some embodiments, the ENPP1 protein binds nucleotide triphosphates (e.g., ATP, UTP, GTP, TTP, and CTP), pNP-TMP, 3',5'-cAMP, and 2'-3'-cGAMP and converts the nucleotide triphosphates to inorganic pyrophosphate [see, e.g., Kato K. et al., Proc Natl Acad Sci USA. 2012; 109(42): 16876-81; Li L, et al. Nat Chem Biol. 2014; 10(12): 1043-8; Jansen S, et al. Structure. 2012; 20(11): 1948-59; and Onyedibe KI, et al. Molecules. 2019; 24(22)].

[0149] As used herein, the term "enzymatically active" or "biologically active" refers to an ENPP1 polypeptide that exhibits pyrophosphatase and / or phosphodiesterase activity (e.g., capable of binding and / or hydrolyzing ATP to AMP and PPi, and / or AP3a to ATP). For example, the pyrophosphatase / phosphodiesterase domain of the ENPP1 protein hydrolyzes extracellular nucleotide triphosphates to generate inorganic pyrophosphate (PPi) and is generally soluble. This activity can be measured using a pNP-TMP assay as previously described (Saunders, et al., 2008, Mol. Cancer Ther. 7(10):3352-62; Albright, et al., 2015, Nat Comm. 6:10006). In certain embodiments, a soluble ENPP1 polypeptide has a pyrophosphatase activity of about 3.4 (±0.4) s ’1 enzyme ’1 k for the substrate ATP cat has a value k catis determined by measuring the rate of hydrolysis of ATP for the polypeptide. In certain embodiments, the soluble ENPP1 polypeptide has a K for the substrate ATP of about 2 pM or less. M has a value of K M is determined by measuring the rate of ATP hydrolysis for the polypeptide. In addition to the teachings herein, these references provide ample guidance for how to generate a soluble ENPP1 protein that retains one or more biological activities (e.g., conversion of a nucleotide to inorganic pyrophosphate).

[0150] In one embodiment, the present disclosure relates to an ENPP1 polypeptide. As described herein, the term soluble ENPP1 polypeptide includes any naturally occurring extracellular domain of the ENPP1 protein, and any variants thereof (including mutants, fragments, and peptidomimetic forms) that retain biological activity (e.g., enzymatic activity). An example of a soluble ENPP1 polypeptide includes, for example, the ENPP1 extracellular domain (SEQ ID NO: 2), as shown in FIG. 3. In certain embodiments, the soluble ENPP1 polypeptide further includes a signal sequence in addition to the extracellular domain of the ENPP1 polypeptide. Exemplary signal sequences include the native signal sequence of the ENPP1 polypeptide, or a signal sequence from another protein, such as the hENPP7 signal sequence. Examples of variant soluble ENPP1 polypeptides are provided in International Patent Application Publication Nos. 2012 / 125182, 2014 / 126965, 2016 / 187408, 2018 / 027024, and 2020 / 047520, which are incorporated by reference in their entireties.

[0151] In some embodiments, the ENPP1 polypeptide is a fusion protein comprising an ENPP1 polypeptide domain and one or more heterologous protein moieties (i.e., polypeptide domains heterologous to ENPP1). An amino acid sequence is understood to be heterologous to ENPP1 if it is not found natively in the form of ENPP1 represented by SEQ ID NO:1. In some embodiments, the heterologous protein moiety comprises an immunoglobulin Fc domain. In some embodiments, the immunoglobulin Fc domain is an IgG1 immunoglobulin Fc domain. In certain embodiments, the soluble ENPP1 polypeptide is C-terminally fused to an Fc domain of human immunoglobulin 1 (IgG1), human immunoglobulin 2 (IgG2), human immunoglobulin 3 (IgG3), and / or human immunoglobulin 4 (IgG4). In other embodiments, the soluble ENPP1 polypeptide is N-terminally fused to an Fc domain of human immunoglobulin 1 (IgG1), human immunoglobulin 2 (IgG2), human immunoglobulin 3 (IgG3), and / or human immunoglobulin 4 (IgG4). In some embodiments, the presence of an Fc domain improves the half-life, solubility, reduces immunogenicity, and increases activity of soluble ENPP1 polypeptides. In certain embodiments, a portion of a native human IgG protein (IgG1, IgG2, IgG3, and IgG4) may be used in place of the Fc portion (e.g., ENPP1-Fc). For example, the present disclosure provides a fusion protein comprising ENPP1 fused to a polypeptide comprising an immunoglobulin constant domain, such as a CH1, CH2, or CH3 domain from human IgG1, IgG2, IgG3, and / or IgG4. The Fc fragment may include a region of a native IgG, such as the hinge region (residues 216-230 of human IgG1 according to the Rabat numbering system), the entire second constant domain CH2 (residues 231-340), and the third constant domain CH3 (residues 341-447).

[0152] As used herein, the term "ENPP1-Fc construct" refers to a soluble form of ENPP1 (e.g., the extracellular domain of ENPP1) recombinantly fused and / or chemically conjugated (including both covalent and non-covalent attachments) to the FcR binding domain of an IgG molecule (preferably human IgG). In certain embodiments, the C-terminus of ENPP1 is fused or conjugated to the N-terminus of the FcR binding domain.

[0153] An example of an amino acid sequence that can be used for the Fc portion of human IgG1 (G1Fc) is SEQ ID NO: 12 (Table 2). In part, the disclosure provides polypeptides comprising, consisting essentially of, or consisting of an amino acid sequence having 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:12.

[0154] In some embodiments, the heterologous protein portion comprises one or more domains selected from the group consisting of polyhistidine, FLAG tag, Glu-Glu, glutathione S-transferase (GST), thioredoxin, protein A, protein G, immunoglobulin heavy chain constant region (Fc), maltose binding protein (MBP), or human serum albumin. The fusion domain may be selected to confer a desired property. For example, some fusion domains are particularly useful for the isolation of fusion proteins by affinity chromatography. For affinity purification purposes, relevant substrates for affinity chromatography are used, such as glutathione, amylase, and nickel or cobalt conjugated resins. Many such substrates are available in "kit" form, such as the QIAexpress™ system (Qiagen) useful with the Pharmacia GST purification system and (HIS6) fusion partners. As another example, the fusion domain may be selected to facilitate detection of the ENPP1 polypeptide. Examples of such detection domains include various fluorescent proteins (e.g., GFP) and "epitope tags," which are usually short peptide sequences for which specific antibodies are available. Well-known epitope tags for which specific monoclonal antibodies are readily available include FLAG, influenza virus hemagglutinin (HA), and c-myc tags. In some cases, the fusion domain has a protease cleavage site, e.g., factor Xa or thrombin, which allows the relevant protease to partially digest the fusion protein, thereby releasing the recombinant protein therefrom. The released protein can then be isolated from the fusion domain by subsequent chromatographic separation.

[0155] In some embodiments, the ENPP1 fusion protein further comprises a linker positioned between the ENPP1 polypeptide domain and one or more heterologous protein moieties (e.g., Fc immunoglobulin domains). In certain embodiments, the soluble ENPP1 polypeptide is directly or indirectly fused to the Fc domain. In some embodiments, the soluble ENPP1 fusion protein comprises a linker between the Fc domain and the ENPP1 polypeptide. In some embodiments, the linker may be an amino acid spacer comprising 1-200 amino acids. Suitable peptide spacers are known in the art and include, for example, peptide linkers comprising flexible amino acid residues such as glycine, alanine, and serine. In some embodiments, the linker comprises a polyglycine linker or a Gly-Ser linker. In some embodiments, the spacer may include a GGGA (SEQ ID NO:21), GGGS (SEQ ID NO:22), GGGG (SEQ ID NO:23), GGGGA (SEQ ID NO:24), GGGGS (SEQ ID NO:25), GGGGG (SEQ ID NO:26), GGAG (SEQ ID NO:27), GGSG (SEQ ID NO:28), AGGG (SEQ ID NO:29), SGGGG (SEQ ID NO:30), SGGG (SEQ ID NO:31), GA (SEQ ID NO:103), GS (SEQ ID NO:104), GG (SEQ ID NO:105), GGA (SEQ ID NO:106), GGS (SEQ ID NO:107), or GGG (SEQ ID NO:108) motif, e.g., multiple or repeated motifs. In some embodiments, the spacer may contain 2 to 12 amino acids including a GA or GS motif, e.g., GA, GS, GAGA (SEQ ID NO: 32), GSGS (SEQ ID NO: 33), GAGAGA (SEQ ID NO: 34), GSGSGS (SEQ ID NO: 35), GAGAGAGA (SEQ ID NO: 36), GSGSGSGS (SEQ ID NO: 37), GAGAGAGAGA (SEQ ID NO: 38), GSGSGSGSGS (SEQ ID NO: 39), GAGAGAGAGAGA (SEQ ID NO: 40), and GSGSGSGSGSGSGS (SEQ ID NO: 41).In some embodiments, the spacer may comprise 3-12 amino acids including a motif of GGA or GGS, e.g., GGA, GGS, GGAGGA (SEQ ID NO: 42), GGSGGS (SEQ ID NO: 43), GGAGGAGGA (SEQ ID NO: 44), GGSGGSGGS (SEQ ID NO: 45), GGAGGAGGAGGA (SEQ ID NO: 46), and GGSGGSGGSGGS (SEQ ID NO: 47). In further embodiments, the spacer may comprise 4-12 amino acids including a motif of GGAG (SEQ ID NO: 48), GGSG (SEQ ID NO: 49), e.g., GGAG (SEQ ID NO: 50), GGSG (SEQ ID NO: 51), GGAGGGAG (SEQ ID NO: 52), GGSGGGSG (SEQ ID NO: 53), GGAGGGAGGGAG (SEQ ID NO: 54), and GGSGGGSGGGSG (SEQ ID NO: 55). In some embodiments, the spacer may comprise the motifs GGGGA (SEQ ID NO: 56) or GGGGS (SEQ ID NO: 57), e.g., GGGAGGGGAGGGGA (SEQ ID NO: 58) and GGGSGGGGSGGGGGS (SEQ ID NO: 59). In some embodiments of the invention, the amino acid spacer between the heterologous protein moiety (e.g., an Fc domain monomer, a wild-type Fc domain, an Fc domain with amino acid substitutions (e.g., one or more substitutions that reduce dimerization), an albumin binding peptide, a fibronectin domain, or human serum albumin) and the soluble ENPP1 polypeptide may be GGG, GGGA (SEQ ID NO: 21), GGGG (SEQ ID NO: 23), GGGAG (SEQ ID NO: 60), GGGAGG (SEQ ID NO: 61), or GGGAGGG (SEQ ID NO: 62).

[0156] In some embodiments, the spacer may also be an amino acid other than glycine, alanine, and serine, such as, for example, TGGGG (SEQ ID NO: 63), AAAL (SEQ ID NO: 64), AAAK (SEQ ID NO: 65), AAAAR (SEQ ID NO: 66), EGKSSGSGSESKST (SEQ ID NO: 67), GSAGSAAGSGEF (SEQ ID NO: 68), AEAAAKEAAAKA (SEQ ID NO: 69), KESGSVSSEQLAQFRSLD (SEQ ID NO: 70), GENLYFQSGG (SEQ ID NO: 71), SACYCELS (SEQ ID NO: 72), RSIAT (SEQ ID NO: 73), RPACKIPNDLKQKVMNH (SEQ ID NO: 74), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG (SEQ ID NO: 75), AAANSSIDLISVPVDSR (SEQ ID NO: 76), GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 77), (R) m ; m = 0-15 (SEQ ID NO: 78), DSSSEEKFLRRIGRFG (SEQ ID NO: 79), EEEEEEEPRGDT (SEQ ID NO: 80), APWHLSSQYSRT (SEQ ID NO: 81), STLPIPHEFSRE (SEQ ID NO: 82), VTKHLNQISQSY (SEQ ID NO: 83), (E) m ; m = 1-15 (SEQ ID NO: 84), RSGSGGS (SEQ ID NO: 85), (D) m ;m=1-15 (SEQ ID NO:86), LVIMSLGLGLGLGLRK (SEQ ID NO:87), VIMSLGLGLGLGLRK (SEQ ID NO:88), IMSLGLGLGLGLRK (SEQ ID NO:89), MSLGLGLGLGLRK (SEQ ID NO:90), SLGLGLGLGLRK (SEQ ID NO:91), LGLGLGLGLRK (SEQ ID NO:92), GLGLGLGLRK (SEQ ID NO:93), LGLGLGLRK (SEQ ID NO:94), GLGLGLRK (SEQ ID NO:95), LGLGLRK (SEQ ID NO:96), GLGLRK (SEQ ID NO:97), LGLRK (SEQ ID NO:98), GLRK (SEQ ID NO:99), (K) m;m=1-15 (SEQ ID NO:100), LIN (SEQ ID NO:109), NSS (SEQ ID NO:110), ESS (SEQ ID NO:111), RQQ (SEQ ID NO:112), KR (SEQ ID NO:113), LRK (SEQ ID NO:114), or RK (SEQ ID NO:115). In some embodiments, the spacer may include a motif, e.g., a multiple or repeating motif, of EAAAK (SEQ ID NO:101). In some embodiments, the spacer may include a motif, e.g., a multiple or repeating motif, of a proline-rich sequence, such as (XP)n, where X can be any amino acid (e.g., A, K, or E) and n is 1 to 5, and PAPAP (SEQ ID NO:102). SEQ ID NO:3 - ENPP2 signal sequence

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[0157] The length of the peptide spacer and the amino acids used can be adjusted depending on the two proteins involved and the degree of flexibility desired in the final protein fusion polypeptide. The length of the spacer can be adjusted to ensure proper protein folding and avoid aggregate formation.

[0158] In some embodiments, the different elements of a fusion protein (e.g., an immunoglobulin Fc fusion protein) may be arranged in any manner consistent with the desired functionality. For example, a soluble ENPP1 polypeptide domain may be arranged C-terminal to a heterologous protein portion, or alternatively, a heterologous protein portion may be arranged C-terminal to a soluble ENPP1 polypeptide domain. The soluble ENPP1 polypeptide domain and the heterologous protein portion may be directly or indirectly linked in a fusion protein, and additional domains or amino acid sequences may be included C- or N-terminal to either domain or between domains. A preferred fusion protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 9-11. In some embodiments, the ENPP1 fusion polypeptide consists of or comprises SEQ ID NO: 9. In some embodiments, the ENPP1 fusion polypeptide consists of or comprises SEQ ID NO: 10. In some embodiments, the ENPP1 fusion polypeptide consists of or comprises SEQ ID NO: 11.

[0159] In some embodiments, the soluble ENPP1 polypeptides of the present disclosure comprise one or more heterologous moieties. Optionally, the soluble ENPP1 polypeptides comprise one or more heterologous moieties selected from glycosylated amino acids, PEGylated amino acids, phamesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids conjugated to lipid moieties, and amino acids conjugated to organic derivatizing agents. In some embodiments, the soluble ENPP1 polypeptides disclosed herein are further modified. Such modifications include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation, and acylation. As a result, the soluble ENPP1 polypeptides may comprise non-amino acid elements, such as polyethylene glycol, lipids, polysaccharides or monosaccharides, and phosphates. The effect of such non-amino acid elements on the functionality of the soluble ENPP1 polypeptides may be tested as described herein for other soluble ENPP1 polypeptides. If the polypeptides of the present disclosure are produced intracellularly by cleaving the nascent form of the polypeptide, post-translational processing may also be important for correct folding and / or function of the protein. Different cells (e.g., CHO, HeLa, MDCK, 293, WI38, NIH-3T3, or HEK293) may be selected to have specific cellular and characteristic mechanisms for such post-translational activity and to ensure correct modification and processing of the soluble ENPP1 polypeptide.

[0160] As used herein, the percentage of "identity" between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be achieved in a variety of ways that are within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, or CLUSTAL OMEGA software. In some embodiments, alignment is performed using CLUSTAL OMEGA software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms required to achieve maximum alignment over the entire length of the sequences being compared.

[0161] In some embodiments, the activity of soluble ENPP1 polypeptides may also be tested in cell-based or in vivo assays. For example, the effect of soluble ENPP1 polypeptides on the production of inorganic pyrophosphate (PPi) can be measured. Specifically, the pyrophosphatase / phosphodiesterase domain of ENPP1 protein hydrolyzes extracellular nucleotide triphosphates to generate inorganic pyrophosphate (PPi), which is generally soluble. This activity can be measured using pNP-TMP assays and HPLC-based ATP hydrolysis assays as previously described (Saunders, et al., 2008, Mol. Cancer Ther. 7(10):3352-62; Albright, et al., 2015, Nat Comm. 6:10006). The effect of soluble ENPP1 polypeptides on the expression of genes involved in ENPP1-related diseases, such as ARHR2 (e.g., transcription of fibroblast growth factor 23 in osteoblasts and osteoclasts), can be evaluated. This may optionally be performed in the presence of one or more nucleotide triphosphates, or other ENPP1 substrates, and the cells may be transfected to produce soluble ENPP1 polypeptides. Similarly, soluble ENPP1 polypeptides may be administered to mice or other animals, and the effect on ENPP1-associated disease may be assessed using art-recognized methods.

[0162] In some embodiments, the ENPP1 polypeptide used according to the methods described herein is an isolated polypeptide. As used herein, an isolated protein or polypeptide is one that is separated from components of its natural environment. In some embodiments, the polypeptide of the present disclosure is purified to greater than 95%, 96%, 97%, 98%, or 99% purity, for example, as determined by electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatographic (e.g., ion exchange or reverse phase HPLC) analysis. Methods for the assessment of purity are well known in the art [see, for example, Flatman et al., (2007) J. Chromatogr. B 848:79-87]. In some embodiments, the soluble ENPP1 polypeptide used according to the methods described herein is a recombinant polypeptide.

[0163] The ENPP1 polypeptides of the present disclosure may be produced by a variety of techniques known in the art. For example, the polypeptides of the present disclosure may be synthesized using standard protein chemistry techniques, such as those described in Bodansky, M. Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) and Grant GA (ed.), Synthetic Peptides: A User's Guide, WH Freeman and Company, New York (1992). In addition, automated peptide synthesizers are commercially available (e.g., Advanced ChemTech Model 396; Milligen / Biosearch 9600). Alternatively, the polypeptides of the present disclosure, including fragments and / or variants thereof, may be produced recombinantly using a variety of expression systems [e.g., E. coli, Chinese hamster ovary (CHO) cells, COS cells, baculovirus, yeast Pichia], as is well known in the art. The proteins may be produced in either adherent or suspension cells. In some embodiments, the fusion proteins are expressed in CHO cells. To establish a stable cell line, the nucleic acid sequence encoding the ENPP1 construct is cloned into a suitable vector for large-scale protein production. In further embodiments, modified or unmodified polypeptides of the present disclosure may be produced by digestion of recombinantly produced full-length ENPP1 polypeptides, for example, by using proteases such as trypsin, thermolysin, chymotrypsin, pepsin, or paired basic amino acid converting enzymes (PACE). Computer analysis (using commercially available software, for example, MacVector, Omega, PCGene, Molecular Simulation, Inc.) may be used to identify protein cleavage sites. Alternatively, such polypeptides may be produced from recombinantly produced full-length ENPP1 polypeptides using chemical cleavage (e.g., cyanogen bromide, hydroxylamine, etc.).

[0164] Many expression systems are known and can be used to produce ENPP1 fusion proteins, including bacteria (e.g., E. coli and Bacillus subtilis), yeast (e.g., Saccharomyces cerevisiae, Kluyveromyces lactis, and Pichia pastoris), filamentous fungi (e.g., Aspergillus), plant cells, animal cells, and insect cells. The desired protein can be produced in a conventional manner, for example, from a coding sequence inserted into the host chromosome or a free plasmid.

[0165] Yeast can be transformed with the coding sequence of the desired protein in any of the usual ways (e.g., electroporation). Methods for transformation of yeast by electroporation are disclosed in Becker & Guarente, 1990, Methods Enzymol. 194:182. Successfully transformed cells, i.e., cells containing the DNA construct of the present disclosure, can be identified by well-known techniques. For example, cells resulting from the introduction of an expression construct can be grown to produce ENPP1 polypeptide. Cells can be harvested, lysed, and their DNA content examined for the presence of DNA using methods such as those described by Southern, 1975, J. Mol. Biol, 98:503 and / or Berent, et al., 1985, Biotech 3:208. Alternatively, the presence of protein in the supernatant can be detected using antibodies.

[0166] Useful yeast plasmid vectors include pRS403-406 and pRS413-416, generally available from Stratagene Cloning Systems (La Jolla, Calif., USA); plasmids pRS403, pRS404, pRS405, and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers I-11S3, TRP1, LEU2, and 1JRA3. Plasmid pRS413-416 is a Yeast Centromere plasmid (YCps).

[0167] Various methods have been developed to operably link DNA to a vector via complementary cohesive ends. For example, complementary homopolymer tracts can be added to the DNA segment to be inserted into the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymer tails to form a recombinant DNA molecule.

[0168] Synthetic linkers containing one or more restriction sites provide an alternative method of joining DNA segments to vectors. DNA segments generated by endonuclease restriction digestion are treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding 3'-single-stranded ends by their 3'-5'-nucleolytic activity and fill in recessed 3'-ends by their polymerization activity.

[0169] Thus, the combination of these activities produces blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that can catalyze the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase. As a result, the products of the reaction are DNA segments carrying polymer linker sequences at their ends. These DNA segments can be cut with appropriate restriction enzymes and ligated into expression vectors that have been cut with enzymes that produce ends that are compatible with those of the DNA segments.

[0170] Then, single stably transfected cell clones are established and screened for high expression clones of desired ENPP1 fusion protein. Screening of single cell clones for ENPP1 protein expression can be achieved in a high-throughput manner in 96-well plates using the synthetic enzyme substrate pNP-TMP as previously described (Albright, et al., 2015, Nat Commun. 6:10006). Upon identification of high expression clones through screening, protein production can be achieved in shake flasks or bioreactors as previously described in Albright, et al., 2015, Nat. Commun. 6:10006.

[0171] Purification of ENPP1 can be achieved using a combination of standard purification techniques known in the art. After purification, ENPP1-Fc is purified by precipitation with ZnCl2 at a concentration of 5-7 mg / ml. 2+ and Mg 2+ The antibody can be dialyzed into PBS supplemented with 0.1% NaCl (PBSplus) and frozen in aliquots of 200-500 pl at -80° C. The aliquots can be thawed immediately prior to use and the specific activity of the solution adjusted to 31.25 au / ml (or approximately 0.7 mg / ml depending on the preparation) by dilution in PBSplus.

[0172] 4. Exemplary Uses In certain aspects, the present disclosure relates to the use of certain soluble ENPP1 polypeptides (e.g., and fusion proteins thereof) to reduce and / or prevent the progression of pathological calcification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the polypeptide fusions and / or soluble polypeptides disclosed herein (e.g., SEQ ID NOs: 2, 9, 10, and 11). In certain embodiments, the pathological calcification is selected from the group consisting of idiopathic infantile arterial calcification (IIAC) and calcification of atherosclerotic plaques. In certain embodiments, the pathological ossification is selected from the group consisting of ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, and osteoarthritis.

[0173] In some embodiments, the present disclosure contemplates a method of reducing and / or preventing the progression of pathological ossification in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a soluble ENPP1 polypeptide and / or an ENPP1 fusion polypeptide disclosed herein (e.g., SEQ ID NOs: 2, 9, 10, and 11).

[0174] In some embodiments, the present disclosure contemplates a method of reducing or preventing the progression of ectopic calcification of soft tissue, including reducing, ameliorating, or preventing vascular calcification, in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a soluble ENPP1 polypeptide or an ENPP1 fusion polypeptide disclosed herein.

[0175] In some embodiments, the present disclosure contemplates a method for reducing or preventing the progression of a disease caused by ENPP1 deficiency (e.g., GACI and ARHR2). ENPP1 deficiency is characterized by a reduced level of ENPP1 activity and / or a lack of expression of ENPP1 level (compared to that of ENPP1 activity level or ENPP1 expression level, respectively, in a healthy subject) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a soluble ENPP1 polypeptide or ENPP1 fusion protein (e.g., SEQ ID NOs: 2, 9, 10, and 11) disclosed herein. In some embodiments, the ENPP1 deficiency is GACI. In some embodiments, the ENPP1 deficiency is ARHR2.

[0176] In some embodiments, the present disclosure contemplates a method of reducing or preventing disease progression caused by lower levels of plasma PPi in a subject in need thereof, the method including administering to the subject a therapeutically effective amount of a polypeptide disclosed herein to increase the subject's plasma PPi to normal or supranormal levels (30-50% higher) and then maintaining the plasma PPi at a constant normal or supranormal level. A normal level of plasma ppi corresponds to 2-5 μM, and in some embodiments, the normal level is 2-3 μM. (Bernhard et al., A Reference range for Plasma levels of Inorganic Pyrophosphate in Children using the ATP Sulfurylase method, Journal of Clinical Endocrinology & Metabolism, 2021) The method further includes administering additional therapeutically effective amounts at intervals of 2 days, 3 days, 1 week, or 1 month to maintain the subject's plasma PPi at a constant normal or supranormal level to reduce or prevent the progression of pathological calcification or ossification. In certain embodiments, the soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein can be used to increase pyrophosphate (PPi) levels in subjects with lower than normal levels. In other embodiments, the soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein can be used to reduce or prevent the progression of pathological calcification or ossification in subjects with lower than normal levels of PPi. In some embodiments, the soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein can be used to treat ENPP1 deficiency (e.g., GACI and ARHR2) manifested by reduced extracellular PPi concentration in a subject. In certain embodiments, the steady-state level of plasma PPi achieved after administration of a first dose of the soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein is maintained for a period of at least 2 days, at least 4 days, at least 1 week, or at least 1 month.

[0177] In some embodiments, the present disclosure contemplates a method of reducing or preventing the progression of a disease caused by lower than normal levels of plasma PPi in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein (e.g., SEQ ID NOs: 2, 9, 10, and 11) to increase and / or maintain the subject's plasma PPi at a level that is about 90%, 95%, 100%, 105%, 110%, 120%, 130%, 140%, or 150% of the normal PPi level. In certain embodiments, the method includes further administering a polypeptide disclosed herein every 2 days, 3 days, 1 week, or 1 month to maintain plasma PPi levels at a level that is about 90%, 95%, 100%, 105%, 110%, 120%, 130%, 140%, or 150% of normal PPi levels, thereby preventing the progression of pathological calcification or ossification.

[0178] In certain embodiments, a second dose of a soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein is administered to the subject after a suitable time interval of about 2 days, about 4 days, about 1 week, or about 1 month such that the steady state level of plasma PPi is maintained at a constant or steady state level and the subject does not revert to the lower level of PPi that they had prior to administration of the first dose of the construct disclosed herein.

[0179] Without intending to be bound by theory, it is believed that maintaining steady state plasma PPi concentrations at normal levels reduces and / or prevents the progression of pathological calcification and pathological ossification in a subject.

[0180] In some embodiments, the present disclosure contemplates a method of treating, reversing, or preventing the progression of ossification of the posterior longitudinal ligament (OPLL) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein (e.g., SEQ ID NOs: 2, 9, 10, and 11).

[0181] In some embodiments, the present disclosure contemplates a method of treating, reversing, or preventing the progression of hypophosphatemic rickets in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a soluble ENPP1 polypeptide or an ENPP1 fusion polypeptide disclosed herein (e.g., SEQ ID NOs: 2, 9, 10, and 11).

[0182] In some embodiments, the soluble ENPP1 polypeptides of the present disclosure treat human or animal disorders or conditions, such as ectopic calcification (e.g., soft tissue calcification, arterial calcification, and vascular calcification), chronic kidney disease (CKD), end stage renal disease (ESRD), uremic arteriolar calcification (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), hypophosphatemic rickets, osteoarthritis, age-related arterial sclerosis, idiopathic infantile arterial calcification (IIAC), calcification of atherosclerotic plaques, disorders associated with ENPP1 deficiency (e.g., autosomal recessive hypophosphatemic rickets type 2 (ARHR2) and infantile generalized arterial calcification (GACI)], pathogenic mutations in the ABCC6 gene (e.g., ABCC6 deficiency), pseudoxanthoma elasticum (PXE), etc.), and pathological ossification. Examples of ENPP1 polypeptides include human ENPP1 precursor polypeptides (eg, SEQ ID NO: 1) and / or soluble human ENPP1 polypeptides (eg, SEQ ID NOs: 2, 9, 10, and 11).

[0183] In other embodiments, the soft tissue comprises an atherosclerotic plaque. In some embodiments, the soft tissue comprises a muscular artery. In some embodiments, the soft tissue is selected from the group consisting of a joint and a spine. In some embodiments, the joint is selected from the group consisting of a joint of the hand and a joint of the foot. In some embodiments, the soft tissue is selected from the group consisting of articular cartilage and spinal disc cartilage. In some embodiments, the soft tissue comprises a blood vessel. In some embodiments, the soft tissue comprises a connective tissue.

[0184] In some embodiments, the present disclosure contemplates a method of treating, reversing, or preventing the progression of pseudoxanthoma elasticum (PXE) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein (e.g., SEQ ID NOs: 2, 9, 10, and 11).

[0185] In some embodiments, the present disclosure contemplates a method of reducing or preventing the progression of age-related arterial stiffening in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a soluble ENPP1 polypeptide or an ENPP1 fusion polypeptide disclosed herein (e.g., SEQ ID NOs: 2, 9, 10, and 11).

[0186] In some embodiments, the present disclosure contemplates a method of treating, reversing, or preventing the progression of atherosclerotic plaque calcification in a vascular artery in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein (e.g., SEQ ID NOs: 2, 9, 10, and 11).

[0187] In some embodiments, the present disclosure contemplates a method of treating, reversing, or preventing the progression of osteoarthritis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a soluble ENPP1 polypeptide or an ENPP1 fusion polypeptide disclosed herein (e.g., SEQ ID NOs: 2, 9, 10, and 11).

[0188] In some embodiments, the present disclosure contemplates a method of treating, reversing, or preventing the progression of arterial sclerosis due to progeria in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein (e.g., SEQ ID NOs: 2, 9, 10, and 11).

[0189] In some embodiments, the present disclosure contemplates a method of treating, reversing, or preventing progression of X-linked hypophosphatemic rickets (XLH), hereditary hypophosphatemic rickets (HHRH), hypophosphatemic bone disease (HBD), ossification of the posterior longitudinal ligament (OPLL), autosomal dominant hypophosphatemic rickets (ADHR), and / or autosomal recessive hypophosphatemic rickets (ARHR2) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein (e.g., SEQ ID NOs: 2, 9, 10, and 11).

[0190] In some embodiments, the present disclosure contemplates a method of treating, reversing, or preventing the progression of age-related bone loss in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a soluble ENPP1 polypeptide or ENPP1 fusion polypeptide disclosed herein (e.g., SEQ ID NOs: 2, 9, 10, and 11).

[0191] In some embodiments, the present disclosure contemplates a method of treating, reversing, or preventing the progression of ankylosing spondylitis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a soluble ENPP1 polypeptide or an ENPP1 fusion polypeptide disclosed herein (e.g., SEQ ID NOs: 2, 9, 10, and 11).

[0192] In some embodiments, the present disclosure contemplates a method of treating, reversing, or preventing the progression of stroke in pediatric sickle cell disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a soluble ENPP1 polypeptide or an ENPP1 fusion polypeptide disclosed herein (e.g., SEQ ID NOs: 2, 9, 10, and 11).

[0193] In certain embodiments, the present disclosure contemplates a method of treating, reversing, or preventing disease progression in a subject diagnosed with progeria, the method comprising administering to the subject a therapeutically effective amount of a soluble ENPP1 polypeptide or an ENPP1 fusion polypeptide disclosed herein (e.g., SEQ ID NOs: 2, 9, 10, and 11).

[0194] In certain embodiments, the polypeptide is a secreted product of an ENPP1 precursor protein expressed in a mammalian cell. In other embodiments, the ENPP1 precursor protein comprises a signal peptide sequence and an ENPP1 polypeptide, and the ENPP1 precursor protein undergoes proteolytic processing into a polypeptide disclosed herein. In some embodiments, in the ENPP1 precursor protein, the signal peptide sequence is conjugated to the N-terminus of the ENPP1 polypeptide. Upon proteolysis, the signal sequence is cleaved from the ENPP1 precursor protein to provide the ENPP1 polypeptide. In certain embodiments, the signal peptide sequence is selected from the group consisting of an ENPP1 signal peptide sequence, an ENPP2 signal peptide sequence, an ENPP7 signal peptide sequence, and an ENPP5 signal peptide sequence.

[0195] In certain embodiments, the polypeptide is administered acutely or chronically to the subject, hi other embodiments, the polypeptide is administered locally, regionally, parenterally, or systemically to the subject.

[0196] In certain embodiments, the subject is a mammal, hi other embodiments, the mammal is a human.

[0197] Those skilled in the art will appreciate that, given the present disclosure, including the methods detailed herein, the present disclosure is not limited to the treatment of a disease or disorder after it has been established. In particular, symptoms of the disease or disorder do not have to be manifested to the extent that they harm the subject, and in fact the disease or disorder does not have to be detected in the subject before treatment is administered. That is, significant pathology from the disease or disorder does not have to occur before the ENPP1 polypeptide of the present invention can provide a benefit.

[0198] In certain aspects, the present disclosure relates to a method for preventing disease and disorders in a subject, in that a soluble ENPP1 polypeptide or an ENPP1 fusion polypeptide disclosed herein may be administered to a subject before the onset of the disease or disorder, thereby preventing the disease or disorder from developing. Thus, the present disclosure relates to a method for preventing or delaying the onset of a disease or disorder in a subject, or reducing its progression or growth, comprising administering an ENPP1 polypeptide to a subject before detection of the disease or disorder. In certain embodiments, an ENPP1 polypeptide is administered to a subject with a strong family history of the disease or disorder, thereby preventing or delaying the onset or progression of the disease or disorder.

[0199] Armed with the disclosure herein, one of skill in the art will therefore understand that preventing a disease or disorder in a subject includes administering an ENPP1 polypeptide to the subject as a preventative measure against the disease or disorder.

[0200] In certain embodiments, the lyophilized formulations disclosed herein may be administered to deliver a dose of 1 ng / kg / day to 100 mg / kg / day. In other embodiments, the lyophilized formulations disclosed herein may be administered to deliver a dose of 1 ng / kg / day to 500 mg / kg / day.

[0201] In some embodiments, the lyophilized formulations disclosed herein may be administered to deliver a dose of 0.1 mg / kg. In some embodiments, the lyophilized formulations disclosed herein may be administered to deliver a dose of 0.2 mg / kg. In some embodiments, the lyophilized formulations disclosed herein may be administered to deliver a dose of 0.3 mg / kg. In some embodiments, the lyophilized formulations disclosed herein may be administered to deliver a dose of 0.4 mg / kg. In some embodiments, the lyophilized formulations disclosed herein may be administered to deliver a dose of 0.5 mg / kg. In other embodiments, the lyophilized formulations disclosed herein may be administered to deliver a dose of 0.6 mg / kg. In other embodiments, the lyophilized formulations disclosed herein may be administered to deliver a dose of 0.7 mg / kg. In other embodiments, the lyophilized formulations disclosed herein may be administered to deliver a dose of 0.8 mg / kg. In other embodiments, the lyophilized formulations disclosed herein may be administered to deliver a dose of 0.9 mg / kg. In other embodiments, the lyophilized formulations disclosed herein may be administered to deliver a dose of 1 mg / kg. In other embodiments, the lyophilized formulations disclosed herein may be administered to deliver a dose of 1.2 mg / kg. In other embodiments, the lyophilized formulations disclosed herein may be administered to deliver a dose of 1.4 mg / kg. In other embodiments, the lyophilized formulations disclosed herein may be administered to deliver a dose of 1.6 mg / kg. In other embodiments, the lyophilized formulations disclosed herein may be administered to deliver a dose of 1.8 mg / kg.

[0202] The relative amounts of active ingredient (e.g., soluble ENPP1 polypeptides and fusion proteins thereof), pharma- ceutically acceptable carrier, and any additional ingredients in the lyophilized formulations disclosed herein will vary depending on the identity, size, and condition of the subject being treated, and further depending on the route by which the composition is administered. By way of example, the compositions may contain from about 0.1% to about 100% (w / w) active ingredient.

[0203] As used herein, a "unit dose" is a discrete amount of a lyophilized formulation containing a predetermined amount of active ingredient (e.g., soluble ENPP1 polypeptide and its fusion protein). The amount of active ingredient is generally equal to the dosage of the active ingredient to be administered to a subject, or a convenient fraction of such a dosage, such as, for example, one-half or one-third of such a dosage. The unit dosage form may be for one of a single daily dose or multiple daily doses (e.g., about 1-4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

[0204] In some embodiments, ENPP1-Fc may be present in the formulation at a concentration ranging from about 0.1 to about 300 mg / ml. In some embodiments, the concentration of ENPP1-Fc is about 0.5 mg / ml, about 1 mg / ml, about 2 mg / ml, about 2.5 mg / ml, about 3 mg / ml, about 3.5 mg / ml, about 4 mg / ml, about 4.5 mg / ml, about 5 mg / ml, about 5.5 mg / ml, about 6 mg / ml, about 6.5 mg / ml, about 7 mg / ml, about 7.5 mg / ml, about 8 mg / ml, about 8.5 mg / ml, about 9 mg / ml, about 9.5 mg / ml, about 10 mg / ml, about 11 mg / ml, about 12 mg / ml, about 13 mg / ml, about 14 mg / ml, about 15 mg / ml, about 16 mg / ml, about 17 mg / ml, about 18 mg / ml, about 19 mg / ml, about 20 mg / ml, about 21 mg / ml, about 22 mg / ml, about 23 mg / ml, about 24 mg / ml, about 25 mg / ml, about 26 mg / ml, about 27 mg / ml, about 28 mg / ml, about 29 mg / ml, about 30 mg / ml, about 31 mg / ml, about 32 mg / ml, about 33 mg / ml, about 34 mg / ml, about 35 mg / ml, about 36 mg / ml, about 37 mg / ml, about 38 mg / ml, about 39 mg / ml, about 40 mg / ml, about 41 mg / ml, about 42 mg / ml, about 43 mg / ml, about 44 mg / ml, about 45 mg / ml, about 46 mg / ml, about 47 mg / ml, about 48 mg / ml, about 4 5mg / ml, about 16mg / ml, about 17mg / ml, about 18mg / ml, about 19mg / ml, about 20mg / ml, about 21mg / ml, about 22mg / ml, about 23mg / ml, about 24mg / ml, about 25mg / ml, about 26mg / ml, about 27mg / ml, about 2 8mg / ml, about 29mg / ml, about 30mg / ml, about 31mg / ml, about 32mg / ml, about 33mg / ml, about 34mg / ml, about 35mg / ml, about 36mg / ml, about 37mg / ml, about 38mg / ml, about 39mg / ml, about 40mg / ml, about 41 mg / ml, about 42 mg / ml, about 43 mg / ml, about 44 mg / ml, about 45 mg / ml, about 46 mg / ml, about 47 mg / ml, about 48 mg / ml, about 49 mg / ml, about 50 mg / ml, about 51 mg / ml, about 52 mg / ml, about 53 mg / ml, about 54 mg / ml, approximately 55 mg / ml, approximately 56 mg / ml, approximately 57 mg / ml, approximately 58 mg / ml, approximately 59 mg / ml, approximately 60 mg / ml, approximately 70 mg / ml, approximately 80 mg / ml, approximately 90 mg / ml, approximately 100 mg / ml, approximately 101 mg / ml, approximately 102 mg / ml, approximately 102.5mg / ml, about 103mg / ml, about 103.5mg / ml, about 104mg / ml, about 104.5mg / ml, about 105mg / ml, about 105.5mg / ml, about 106mg / ml, about 106.5mg / ml, about 107mg / ml, about 107.5mg / ml, approximately 108mg / ml, approximately 108.5mg / ml, approximately 109mg / ml, approximately 109.5mg / ml, approximately 110mg / ml, approximately 111mg / ml, approximately 112mg / ml, approximately 113mg / ml, approximately 114mg / ml, approximately 115mg / ml, approximately 116mg / ml,About 117 mg / ml, about 118 mg / ml, about 119 mg / ml, about 120 mg / ml, about 121 mg / ml, about 122 mg / ml, about 123 mg / ml, about 124 mg / ml, about 125 mg / ml, about 126 mg / ml, about 127 mg / ml, about 128 mg / ml, about 129 mg / ml, about 130 mg / ml, about 131 mg / ml, about 132 mg / ml, about 133 mg / ml, about 134 mg / ml, about 135 mg / ml, about 136 mg / ml, about 137 mg / ml, about 138 mg / ml, about 139 mg / ml, about 140 mg / ml, about 141 mg / ml, about 142 mg / ml, about 143 mg / ml, about 144 mg / ml, about 145 mg / ml, about 146 mg / ml, about 147 mg / ml, about 148 mg / ml, about 149 mg / ml, about 150 mg / ml, about 151 mg / ml, about 152 mg / ml, about 153 mg / ml, about 154 mg / ml, about 155 mg / ml, about 156 mg / ml, about 157 mg / ml, about 158 mg / ml, about 159 mg / ml, about 160 mg / ml, about 170 mg / ml, about 180 mg / ml, about 190 mg / ml, about 200 mg / ml, about 201 mg / ml, about 202 mg / ml, about 203 mg / ml, about 204 mg / ml, about 205 mg / ml, about 206 mg / ml, about 207 mg / ml, about 208 mg / ml, about 209 mg / ml, about 210 mg / ml, about 211 mg / ml, about 212 mg / ml, about 213 mg / ml, about 214 mg / ml, about 215 mg / ml, about 216 mg / ml, about 217 mg / ml, about 218 mg / ml, about 219 mg / ml, about 220 mg / ml, about 221 mg / ml, about 222 mg / ml, about 223 mg / ml, about 224 mg / ml, about 225 mg / ml, about 226 mg / ml, about 227 mg / ml, about 228 mg / ml, about 229 mg / ml, about 230 mg / ml, about 231 mg / ml, about 232 mg / ml, about 233 mg / ml, about 234 mg / ml, about 235 mg / ml, about 236 mg / ml, about 237 mg / ml, about 238 mg / ml, about 239 mg / ml, about 240 mg / ml, about 241 mg / ml, about 242 mg / ml, about 243 mg / ml, about 244 mg / ml, about 245 mg / ml, about 246 mg / ml, about 247 mg / ml, about 248 mg / ml, about 249 mg / ml, about 250 mg / ml, about 251 mg / ml, about 252 mg / ml,about 253 mg / ml, about 254 mg / ml, about 255 mg / ml, about 256 mg / ml, about 257 mg / ml, about 258 mg / ml, about 259 mg / ml, about 260 mg / ml, about 270 mg / ml, about 280 mg / ml, about 290 mg / ml, or about 300 mg / ml.

[0205] The regimen of administration can affect what constitutes an effective amount. For example, several divided doses and staggered doses can be administered daily or sequentially, or doses can be continuously infused or bolus injected. Further, the dosage of the therapeutic formulation can be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0206] Administration of compositions of the present disclosure (e.g., soluble ENPP1 polypeptides as well as fusion proteins thereof) to a patient, such as a mammal (i.e., human), may be carried out using known procedures at dosages and for periods of time effective to treat the patient's disease or disorder. The effective amount of therapeutic compound required to achieve a therapeutic effect may vary according to factors such as the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of treatment, other drugs, compounds, and materials used in combination with the compound, the state of the disease or disorder of the patient being treated, the age, sex, weight, condition, general health, and medical history, and similar factors well known in the medical arts. Dosage regimens may be adjusted to provide an optimal therapeutic response. Dosage is determined based on the biological activity of the therapeutic compound, which in turn depends on the half-life and plasma area under time of the therapeutic compound curve. Polypeptides according to the present disclosure are administered at appropriate time intervals, such as every 2 days, or every 4 days, or weekly, or monthly, to achieve continuous levels of plasma PPi that are either close to normal (1-3 pM) levels or above normal levels (30-50% higher). The therapeutic dosage of the ENPP1 polypeptide may also be determined based on the half-life or the rate at which the therapeutic polypeptide is excreted from the body. The polypeptide according to the present disclosure is administered at appropriate time intervals, either every 2 days, or every 4 days, weekly, or monthly, to achieve a constant level of ENPP1 enzyme activity.

[0207] For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range of the therapeutic compounds disclosed herein is about 0.01-50 mg / kg body weight / day. In some embodiments, the effective dose range of the therapeutic compounds disclosed herein is about 50 ng-500 ng / kg body weight, preferably 100 ng-300 ng / kg body weight. One of ordinary skill in the art would be able to examine the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

[0208] Soluble ENPP1 polypeptides and fusion proteins thereof may be administered to patients as frequently as several times a day, or less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every few months or once a year or less. It is understood that the amount of soluble ENPP1 polypeptides and fusion proteins thereof administered per day may be administered, in non-limiting examples, every day, every other day, every second day, every third day, every fourth day, or every fifth day. For example, every other day administration may involve a 5 mg per day dose starting on Monday, a first subsequent 5 mg per day dose on Wednesday, a second subsequent 5 mg per day dose on Friday, etc. The frequency of dosing is readily apparent to those skilled in the art and depends on any number of factors, including, but not limited to, the type and severity of the disease being treated, and the type and age of the patient.

[0209] The actual dosage levels of the active ingredients (e.g., soluble ENPP1 polypeptides and fusion proteins thereof) in the lyophilized formulations of the present disclosure may be varied to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient.

[0210] A medical practitioner, e.g., a physician, having ordinary skill in the art, can readily determine and prescribe the effective amount of lyophilized formulation required. For example, the physician or veterinarian can begin dosing with the compounds disclosed herein used in the lyophilized formulation at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0211] In certain embodiments, the compositions disclosed herein are administered to a patient in a dosage range ranging from 1 to 5 or more times per day. In other embodiments, the compositions disclosed herein are administered to a patient in a dosage range including, but not limited to, once per day, once every 2 days, once every 3 days to once per week, and once every 2 weeks. The frequency of administration of the various combination compositions disclosed herein will vary from subject to subject depending on many factors including, but not limited to, age, disease or disorder being treated, sex, overall health, and other factors. Thus, the present disclosure should not be construed as being limited to any particular dosage regime, and the exact dosage and composition administered to any patient will be determined by the attending physician, taking into account all other factors related to the patient.

[0212] In certain embodiments, the present disclosure is directed to a packaged lyophilized formulation comprising a container holding a therapeutically effective amount of a compound disclosed herein, alone or in combination with a second pharmaceutical agent, and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient.

[0213] In certain embodiments, the polypeptide, or precursor protein thereof, is administered to a subject acutely or chronically. In other embodiments, the polypeptide, or precursor protein thereof, is administered to a subject locally, regionally, or systemically. In yet other embodiments, the polypeptide, or precursor protein thereof, is delivered on an encoded vector, which encodes a protein that is transcribed and translated from the vector upon administration of the vector to a subject.

[0214] As used herein, "parenteral administration" of a lyophilized formulation includes any administration route characterized by physical intrusion of the subject's tissue and administration of the lyophilized formulation through an intrusion in the tissue. Thus, parenteral administration includes, but is not limited to, administration of the lyophilized formulation by injection of the composition, application of the composition through a surgical incision, application of the composition through a tissue membrane-permeable non-surgical wound, and the like. In particular, parenteral administration is intended to include, but is not limited to, subcutaneous, intravenous, intraperitoneal, intramuscular, intrasternal injection, and renal dialysis infusion techniques.

[0215] A lyophilized formulation suitable for parenteral administration may comprise one or more ENPP1 polypeptides in combination with one or more pharma- ceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions or emulsions, or sterile powders that can be reconstituted immediately before use into sterile injectable solutions or dispersions, and may contain antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the intended recipient, or suspending or thickening agents. Examples of suitable aqueous and non-aqueous carriers that may be employed in the lyophilized formulations of the present disclosure include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0216] The compositions and formulations may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.

[0217] 5. Arrays [Table 2-1] [Table 2-2] [Table 2-3]

[0218] The terms used herein generally have their ordinary meaning in the art, within the context of this disclosure, and within the specific context in which each term is used. Certain terms are discussed below or elsewhere in this specification to provide additional guidance to the practitioner in describing the compositions and methods of the present disclosure, and how to make and use them. The scope or meaning of any use of a term will be clear from the specific context in which it is used.

[0219] "About" and "approximately" are intended to mean an acceptable degree of error for the quantity measured, generally given the nature or precision of the measurement. Typically, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values.

[0220] Alternatively, particularly in biological systems, the terms "about" and "approximately" may refer to values ​​that are within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold of a given value. Numerical values ​​given herein are approximate unless otherwise stated, meaning that the term "about" or "approximately" can be deduced when not explicitly stated.

[0221] The terms "a" and "an" include plural references unless the context in which the term is used clearly dictates otherwise. The terms "a" (or "an"), as well as "one or more" and "at least one" may be used interchangeably herein. Furthermore, as used herein, "and / or" should be interpreted as a specific disclosure of each of two or more specified features or components, regardless of the presence or absence of the others. Thus, the term "and / or" used in phrases such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (single), and "B" (single). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0222] The numerical ranges disclosed herein are inclusive of the numbers defining the range.

[0223] The polypeptides disclosed herein may include amino acid sequences that are not naturally occurring. Such variants necessarily have less than 100% sequence identity or similarity with the starting molecule. In certain embodiments, the variants will have an amino acid sequence that is less than about 75%-100%, more preferably less than about 80%-100%, more preferably less than about 85%-100%, more preferably less than about 90%-100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%), and most preferably less than about 95%-100% amino acid sequence identity or similarity with the amino acid sequence of the starting (e.g., naturally occurring or wild-type) polypeptide, for example, over the length of the variant molecule.

[0224] Preferred methods and materials are described herein, although methods and materials similar or equivalent to those disclosed herein can also be used in the practice or testing of the disclosed methods and compositions. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. EXAMPLES

[0225] The present invention will now be generally described, which will be more readily understood by reference to the following examples, which are included merely for the purpose of illustrating certain particular embodiments and aspects of the invention, and are not intended to limit the invention.

[0226] Example 1. Generation of ENPP1 fusion proteins The soluble ENPP1 fusion protein was fused to a human Fc domain via a linker (containing leucine, isoleucine, and asparagine) and is hereafter referred to as ENPP1-Fc. Three ENPP1-Fc constructs are shown in Table 2 as SEQ ID NOs: 9, 10, and 11, which were purified from CHO cells.

[0227] Purification of ENPP1-Fc can be achieved by a series of column chromatography steps, including, for example, three or more of protein A chromatography, Q Sepharose chromatography, phenyl sepharose chromatography, size exclusion chromatography, and cation exchange chromatography, in any order. Purification can be completed with viral filtration and buffer exchange. After purification of the protein, the catalytic activity of the ENPP1-Fc protein can be evaluated using pNP-TMP as a chromogenic substrate. Multiple buffer types, pH conditions, excipients, and surfactant concentrations were evaluated in an iterative manner over the course of baseline buffer, additive, solubility, surfactant screening, and cofactor screening to identify formulations that may provide optimal chemical, physical, and structural stability of ENPP1-Fc under both stressed and unstressed conditions.

[0228] Example 2. Production of lyophilized ENPP1 polypeptide formulations Lyophilized ENPP1 polypeptide formulations disclosed herein were produced according to the following steps: ENPP1-Fc at a concentration of 44 mg / mL was buffer exchanged into formulation buffer lacking surfactant. The buffer exchange process was performed for a total of 4 cycles and a total of more than 400-fold dilution. Protein concentration in the buffer-exchanged samples was measured by UV-visible spectroscopy using an extinction coefficient of 1.47 ml / mg*cm and a path length of 1.0 cm. Surfactant (PS20) was added to all formulations to a final concentration of 0.05% during normalization to 50.0 mg / mL. After buffer exchange, the formulations were sterile filtered and filled into triple-rinsed autoclave vials with a fill volume of 0.5 mL and fitted with sterile rubber stoppers.

[0229] The ENPP1-Fc lyophilization cycle (Table 3) consisted of an initial annealing step, where the temperature was cycled from -50°C to -20°C for several hours to allow complete crystallization of the mannitol-containing formulation. After the annealing step, primary drying was initiated by setting the pressure at 100 mTorr and increasing the shelf temperature to 25°C, and the cycle was held for 1793 minutes (approximately 30 hours) at primary drying. After primary drying was completed, secondary drying was performed to remove any residual moisture by increasing the temperature to 40°C and holding for 600 minutes (10 hours). The total duration of the lyophilization cycle was 3648 minutes (approximately 2.5 days). Upon completion of the cycle, the vials were backfilled with nitrogen at a partial pressure of approximately 570 Torr, stoppered, and properly sealed. All lyophilized cakes containing ENPP1-Fc showed intact structure with an ivory color. After reconstitution, the liquid appearance was observed to be brownish yellow, clear, and free of visible particles for all sample types and conditions. Multiple buffers, pH conditions, excipients, additives, surfactants, and cofactors were evaluated in an iterative manner to identify formulations that could provide optimal chemical, physical, and structural stability of ENPP1-Fc under both stressed and unstressed conditions. [Table 3]

[0230] Example 3. Reconstitution of lyophilized ENPP1 polypeptide formulations Reconstitution of the lyophilized samples was evaluated upon reconstitution with 0.5 mL of water for injection. The time until all particulate matter was dissolved was measured. Each lyophilized sample completely dissolved within a period of at least 46 seconds without leaving any visible residue or undissolved material. A summary of the reconstitution data is shown in Table 4. [Table 4]

[0231] Example 4. Assessment of residual moisture in lyophilized ENPP1 polypeptide formulations Residual moisture was assessed for the lyophilized cakes. All lyophilized samples had very low or no moisture content across time points and conditions. Residual moisture content ranged from 0.0% to 0.3%. Summary data is shown in Table 5. [Table 5]

[0232] Example 5: Baseline Buffer Evaluation The stability of ENPP1 polypeptide formulations was evaluated over a pH range of 5.0-8.0 using a panel of candidate buffers including citrate, histidine, phosphate, acetate, sodium bicarbonate, succinate, glycylglycine, and Tris. To evaluate the self-buffering capacity of these formulations, samples were buffer exchanged into water. In this study, ENPP1-Fc was buffer exchanged into 20 mM acetate (pH 5.0), succinate (pH 5.0 and 6.0), citrate (pH 5.0, 6.0, and 7.0), sodium bicarbonate (pH 6.0 and 7.0), histidine (pH 6.0 and 7.0), potassium phosphate (pH 7.0 and 8.0), Tris (pH 7.0 and 8.0), and glycylglycine (pH 8.0) buffers. ENPP1-Fc was buffer exchanged into one of the formulation buffers identified above and concentrated to 2 mg / mL in 0.5 mL. Protein recovery after concentration and buffer exchange was generally comparable across formulations, ranging from approximately 70-100%, with the exception of ENPP1-Fc buffer exchanged into water. Buffer exchanged ENPP1-Fc in water showed 34% recovery, indicating that ENPP1-Fc lacks self-buffering capacity.

[0233] The thermal stability of ENPP1 polypeptide formulations was monitored by differential scanning fluorimetry (DSF), which provides data on the melting temperature (Tm) of the polypeptide. Tm data was collected for 2 mg / mL protein samples. Samples were equilibrated at 20°C for 30 seconds and the temperature was increased to 95°C at a rate of 0.5°C / min while the centroid mean (BCM) of the intrinsic fluorescence spectrum from 250 to 500 nm (266 nm excitation wavelength) was monitored. The inflection point of the BCM vs. temperature curve during the unfolding event (identified by the maximum or minimum of the derivative trace) was identified as the Tm of that transition. Static light scattering (SLS) intensity at 266 nm and 473 nm was also measured in parallel with the DSF measurements to observe the onset temperatures of small and large aggregate formation (Tagg), respectively. Relative thermal stability was affected by both buffer type and formulation pH. Citrate pH 7.0, phosphate pH 7.0 / 8.0, sodium bicarbonate, and glycylglycine samples did not show significant light scattering at 266 nm or 473 nm, indicating a lack of apparent aggregation for these formulations. Acetate, succinate pH 5.0, citrate pH 5.0, and Tris pH 7.0 formulations exhibited the highest Tagg across formulations. For citrate, histidine, and Tris samples, Tagg decreased with increasing pH, with phosphate Tagg appearing to be insensitive to changes in pH. The Tm values ​​surprisingly showed clear trends with buffer type for each pH, ​​indicating better thermal stability for succinate at pH 5.0 and pH 6.0, and phosphate at pH 5.0 and pH 6.0. Summary DSF data is shown in Table 6. [Table 6]

[0234] Size exclusion chromatography (SEC) was used to assess the amount of aggregates and degradation products present in ENPP1 polypeptide formulation samples. The abundance of total high molecular weight (HMW) species, low molecular weight (LMW) species, and major peak purity are reported. SEC results are summarized in Table 7 and Figure 5. All samples were incubated at 30 °C for 48 h prior to analysis by SEC. HMW species were present in all formulations ranging from 3.3 to 14.5%. The lowest levels of HMW species were observed in sodium bicarbonate and phosphate formulations. Higher order HMW species were highest in histidine pH 7.0. [Table 7]

[0235] In summary, the baseline buffer evaluation data indicates that buffer formulations including pH 5.0 or less and pH 6.0 or more provide the greatest stability for the lyophilized ENPP1 polypeptide formulation. Histidine pH 7.0 is less preferred than other buffer systems tested. Acetate is unable to buffer the lyophilized formulation to the required extent. Based on the above data, the buffers were binned into two regimes including those supporting a pH range of pH 6 to pH 7 (succinate, citrate, bicarbonate) and those supporting a pH range of pH 7 to pH 8 (phosphate, Tris, glycylglycine). In the first buffer regime, the citrate formulation exhibited superior Tonset (around 5°C). In the second buffer regime, the Tris formulation exhibited superior Tonset (around 2°C). The pH levels selected for further formulation development were pH 6.0, pH 7.0, and pH 8.0 using the succinate, citrate, and phosphate buffer systems, respectively. For each buffer type, five different pharma- ceutically acceptable excipients were evaluated: arginine, NaCl, sucrose, mannitol, and glycine. The proposed excipients cover a variety of molecular classes (i.e., amino acids, salts, sugars, and polyols).

[0236] Example 6: Pharmaceutically acceptable additive screening The stability of lyophilized ENPP1 polypeptide formulations was evaluated in the presence of various additives with reported stabilizing properties at near isotonic concentrations using the buffer systems defined in the baseline buffer evaluation (20 mM succinate pH 6.0, citrate pH 7.0, and phosphate pH 8.0). ENPP1-Fc was buffer exchanged into each candidate buffer containing either 150 mM NaCl, 150 mM L-arginine, 250 mM sucrose, 250 mM mannitol, or 200 mM glycine.

[0237] DSF / SLS data for formulations of ENPP1-Fc containing additives showed that the additives did not have a large effect on thermal stability as assessed by Tm (DSF) and Tagg (SLS) at both 266 nm and 473 nm. Overall, the DSF data showed comparable melting temperatures and a clear lack of larger aggregates for the majority of the formulations.

[0238] The SEC data (summarized in Table 8 and Figure 6) showed that glycine and NaCl / arginine combinations produced the highest % HMW species among the formulations. NaCl at pH 6.0 and pH 7.0, and arginine pH 6.0 behaved similarly to sucrose. Sucrose produced the lowest % HMW species (5.3% or less) across each of the formulations tested. The SEC data indicates that the formulation containing sucrose provided the highest overall stability. [Table 8]

[0239] Example 7: Solubility screening of lyophilized ENPP1 polypeptide formulations To establish conditions for maximum solubility of lyophilized ENPP1 polypeptide formulations, 20 mM succinate pH 6.0, citrate pH 5.0 and pH 6.0, sodium bicarbonate pH 7.0, and histidine pH 6.0 were used in the presence of isotonic concentrations of sucrose, proline, and NaCl. After buffer exchange and concentration, the samples were divided into two equal volume aliquots, one of which was heat stressed at 40° C. for about 7 days and the other was kept at 5° C. The heat stressed and 5° C. samples were evaluated by SEC for enzyme activity and formation of HMW species. The enzyme assay used to evaluate the activity of each sample was performed by mixing ENPP1-Fc with pNP-TMP. Product formation (pNP) is monitored spectrophotometrically at 405 nm for a period of 5 minutes at 25° C. The specific activity of ENPP1-Fc is calculated by interpolating the change in absorbance over time obtained for each sample preparation to a standard curve of pNP of known concentration. Results are reported relative to the reference sample material, with units corresponding to moles of product produced over time (min). The results of the solubility screening enzyme activity assay are shown in Table 9 and Figure 7. The formulations containing pH 6.0 showed increased thermal stability as shown by the increased enzyme activity observed for each of these samples. Values ​​for pH 5.0 citrate incubated at 40°C were not determined due to gelling of the sample. Overall, the enzyme activity suggests that succinate and citrate at pH 6.0 are the best performing buffer types across the additives evaluated when comparing 5°C vs. 40°C incubated samples. [Table 9]

[0240] The formation of HMW species determined by SEC was consistent with the trends observed in the enzyme activity screen. Succinate and citrate at pH 6.0 exhibited the lowest HMW% regardless of the excipients used in the formulation. Citric acid pH 6.0 at 40°C outperformed all other 40°C samples. Proline and NaCl in citrate pH 5.0 and sodium bicarbonate pH 7.0 yielded the highest HMW species% overall. A summary of the SEC solubility screening data is shown in Table 10 and Figure 8. [Table 10]

[0241] Example 8: Surfactant Screening To identify superior surfactants for use in lyophilized ENPP1 polypeptide formulations, ENPP1-Fc was buffer exchanged into three base formulations (succinate / sucrose pH 6.0, succinate / NaCl pH 6.0, and citrate / sucrose pH 7.0) and a control formulation (histidine / arginine / NaCl pH 6.0). Formulation samples were subjected to stress via freeze-thaw cycles and mechanical stress, plus two small aliquots were reserved as unstressed controls. Freeze-thaw cycle samples were frozen at -80°C for ≥60 min and thawed at room temperature. This process was repeated for a total of five cycles. For agitation stress, samples were placed at room temperature for approximately 72 h, protected from light, on a microplate shaker set at approximately 600 rpm.

[0242] Enzyme activity was measured and SEC-HPLC was performed to assess the stability / aggregation of stressed samples. Enzyme activity between surfactants for all stress conditions and formulations was comparable, indicating that the enzymatic activity of ENPP1-Fc was not affected under the test conditions. SEC evaluation of surfactant testing is shown in Table 11, Figure 9A, and Figure 9B. HMW content % across all formulations indicates that surfactants did not affect samples subjected to freeze / thaw stress, as values ​​are shown to be comparable between stressed samples and their respective controls. For samples subjected to agitation stress, the P188 sample was the most stable of the three tested surfactants. PS20 provided superior stability under agitation stress, producing a decrease in HMW content % across the three surfactants tested. These data indicate that the presence and type of surfactant impacts ENPP1-Fc stability after physical stress. SEC indicates that PS20 is the optimal surfactant type for EnPP1-Fc stability at 50 mg / mL. After agitation, P188 showed increased total levels of higher order aggregates in most excipient / buffer type combinations. [Table 11-1] [Table 11-2]

[0243] Example 9: Cofactor Screening Eleven different cofactor combinations (including a negative control without cofactor) in 20 mM succinate, 250 mM sucrose, pH 6.0 were investigated. Cofactors tested included Zn+2 and Ca+2 with chloride and sulfate as counterions, and adenosine monophosphate (AMP). SEC experiments were performed for each cofactor combination. The SEC data are summarized in Table 12. Figure 10. These data show that AMP / ZnCl exhibited the lowest HMW% levels. However, for the cofactors tested, AMP, Ca2+ with either chloride or sulfate as counterions produced the lowest HMW content%. The Ca2+ cofactor surprisingly stabilized ENPP1-Fc, but also showed the ability to reverse higher order aggregate species that may have formed during sample preparation. These effects are observed by comparing formulations A and C (Figure 11). Zinc produced the highest levels of % HMW content, but the % HMW in the presence of zinc was significantly reduced by the addition of amp, reducing all samples below the % HMW levels of the raw material used to generate Formulation A. These results indicate that the presence and type of cofactor surprisingly and significantly increases the stability of ENPP1-Fc. [Table 12]

[0244] A total of 15 different buffer / cofactor combinations were further investigated. ENPP1-Fc was buffer exchanged into 12 different formulations. Protein stability in the various formulations was evaluated by SEC. Samples were stored at 40° C. for 6 days prior to SEC analysis. Control samples of each formulation were kept at 5° C. for the duration of the study. SEC data are summarized in Table 13 and Figures 12A-B. Consistent with the cofactor data presented above, Ca+2 and Ca+2 / adenosine monophosphate produced the least amount of higher order aggregates across the samples tested. When combined with citrate and sucrose, Ca+2 and Ca+2 / adenosine monophosphate produced the lowest percentage of HMW (8.6%), respectively, indicating that Ca+2 and Ca+2 / adenosine monophosphate are superior cofactors and surprisingly enhance the overall stability of the formulation when combined with citrate and sucrose. [Table 13]

[0245] Example 10: Characterization of preferred lyophilized ENPP1 polypeptide formulations Based on the characterization and stability data presented above, preferred lyophilized ENPP1 polypeptide formulations include succinate and citrate. These preferred formulations are listed in Table 13 and further characterized for polypeptide stability over time. The pH was determined to be pH 6.3±0.1 for each formulation sample containing citrate and pH 6.1±0.1 for each sample containing succinate. Osmolality was measured for ENPP1-Fc samples containing succinate or citrate, and osmolality values ​​for all samples ranged from 265 to 432 mOsm / kg. Summary data is shown in FIG. 13. The main peak purity and higher order aggregates of ENPP1-Fc in these formulations were evaluated by SEC. The results are shown in FIG. 14. Lyophilized ENPP1-Fc in formulations A, B, C, E, and F and the control exhibited relative main peak purity of greater than 94% across all time points and conditions. At 13 weeks stored at 5°C, the succinate samples exhibited a 1% increase in HMW content, while the citrate samples showed an increase of approximately 0.5% in HMW content. Particle size distribution and morphology were assessed by microflow imaging analysis (MFI). In MFI, bright field microscopic images are captured in successive frames as a continuous sample stream passes through a flow cell positioned in the field of view of the microscope system. Images of particles present in the sample are processed by image morphology analysis software that allows for quantification of size and count. Representative images and particle concentrations (per mL) were reported for Equivalent Circle Diameter (ECD) size bins of ≥2 μm, ≥5 μm, ≥10 μm, and ≥25 μm. The ECD of an object is expressed in microns and represents the diameter of a sphere that occupies the same two-dimensional surface area as the particle. The results are summarized in Table 14. Total particle counts were generally within acceptable limits for all formulations. The total particle counts for the 10 μm and larger ECD bins are all less than 749, and the total particle counts for the 25 μm and larger ECD bins are all less than 132, indicating that the formulations disclosed herein are devoid of larger sized particles. [Table 14]

[0246] Based on the data reported above, 20 mM citrate, 88 mM sucrose, 82 mM mannitol, 2 mM calcium chloride, 0.05% PS20 pH 6.3 (formulation A) and 20 mM citrate, 263 mM sucrose, 2 mM calcium chloride, 0.05% PS20 pH 6.3 (formulation B) were selected as the most suitable formulations for lyophilized ENPP1-Fc. These formulations were further investigated for their long-term effect on the stability of the lyophilized polypeptide at various storage conditions. The two formulations were tested at three different storage conditions, 5°C, 25°C, and 40°C, over a period of three months. After three months, all lyophilized cakes containing ENPP1-Fc showed an intact structure with an ivory color. After reconstitution, the appearance of the liquid was observed to be brownish yellow, clear, and free of visible particles for all sample types. Reconstitution occurred within a window consistent with the data described above, with complete reconstitution of the lyophilized samples occurring within 59-108 seconds. The moisture content of both formulations across temperature conditions over time also remained consistent with the moisture content data described above. All lyophilized sample time points at all of the storage temperatures tested had very low or no moisture content across time points and conditions, with residual moisture content ranging from 0.0% to 0.3%. The pH value of each sample at each storage condition also remained consistent over time, with sample pH remaining at pH 6.3±0.1 over the course of three months. Protein recovery relative to a normalized starting concentration of 50 mg / mL was also measured for each sample. Summary protein recovery data is shown in Table 15. Recovery of each sample was consistent with the recovery data for the zero time point positive control. The zero time point positive control was measured to have a concentration of 40.1 mg / mL. All recovered samples showed concentrations that were either within 0.3 mg / mL below the positive control sample or within 8.8 mg / mL above the concentration of the positive control sample. These data indicate that protein recovery does not deteriorate over time under storage conditions of 5° C., 25° C., and 40° C. [Table 15]

[0247] SEC experiments were performed to characterize the tendency of HMW species to develop over a three-month period when samples of the preferred formulations were stored at 5°C, 25°C, and 40°C. A summary of the SEC data for the abundance of total HMW species, LMW species, and major peak species is reported in Table 16. Figure 15 shows a summary of the SEC data for the HMW abundance and major peak abundance over time by storage condition. The positive control zero time point sample had an HMW content of approximately 4%. All preferred formulation samples remained consistent with the control, with an HMW content of ±5% over a three-month period under storage conditions of 5°C, 25°C, and 40°C, respectively. These results show that each preferred formulation can surprisingly prevent the formation of high molecular weight aggregate species over an extended period of at least three months. These data also show that the preferred formulations can stabilize lyophilized polypeptides against the formation of HMW species even when stored at higher or non-refrigerated temperatures such as 25°C and 40°C. [Table 16]

[0248] Specific activity for all preferred formulation samples corresponding to each time point and storage condition was evaluated using the enzyme activity screen described above. Summary data is shown in Table 17. Activity data was plotted over time for each sample, including the frozen control. A representative activity vs. time plot is shown in Figure 16. All samples across time points and storage conditions showed levels of enzyme activity consistent with the positive control, with activity levels of 46009 μmol min -1 mg -1 ~73173μmol min -1 mg -1 The range was. [Table 17]

[0249] The particle size distribution and morphology of all preferred formulation timepoint samples were evaluated by MFI analysis. Representative images and particle concentrations (per mL) were reported for ECD size bins of 2 μm or more, 5 μm or more, 10 μm or more, and 25 μm or more. The results are summarized in Table 18. The total particle counts were surprisingly much lower than those observed in the MFI results above and were within acceptable limits for all preferred formulation samples. The total particle counts for the 10 μm or more ECD bins were all less than 146, and the total particle counts for the 25 μm or more ECD bins were all less than 21, indicating that the preferred formulations disclosed herein can reduce larger particle sizes by almost 10-fold compared to other formulation combinations described in previous MFI analysis. [Table 18]

[0250] The above formulation development studies focused on identifying formulation components that could provide optimal chemical, physical, and conformational stability of the ENPP1-Fc formulation under both stressed and unstressed conditions. Multiple buffer types, pH conditions, excipients, and surfactant concentrations were evaluated iteratively over the course of baseline buffer, excipient, solubility, surfactant, and cofactor testing. Based on the results of the lyophilized formulation testing disclosed herein, 20 mM citrate pH 6.3, 88 mM sucrose, 82 mM mannitol, 2 mM calcium chloride, 0.05% PS20, and 20 mM citrate pH 6.3, 263 mM sucrose, 2 mM calcium chloride, 0.05% PS20 were selected as the most suitable formulations for ENPP1-Fc. These preferred formulations confer maximum chemical, physical, and conformational stability to ENPP1-Fc at a target concentration of 50 mg / mL lyophilized polypeptide formulation.

[0251] In addition, another exemplary formulation (Formulation C1) was tested: 50 mg / mL ENPP1 polypeptide, 20 mM citrate at approximately pH 6.3, 2 mM calcium chloride, 175 mM sucrose, 82 mM (D) mannitol, and 0.05% w / v polysorbate 20. This formulation was investigated for its long-term effect on lyophilized ENPP1-Fc polypeptide stability at various storage conditions over a period of 12 months at three different storage conditions: 5° C., 25° C., and 40° C. After three month increments, the lyophilized cakes containing ENPP1-Fc showed an intact structure with an ivory color. After reconstitution, the liquid appearance was observed to be brownish yellow, clear, and free of visible particles for all sample types. Reconstitution occurred within a window consistent with the data described above, with complete reconstitution of the lyophilized samples occurring within 2-3 minutes. The water content over time and temperature conditions also remained consistent. All lyophilized sample time points at all of the storage temperatures tested had very low or no moisture content across time points and conditions, with residual moisture content ranging from 0.0% to 0.16%. The pH value of each sample at each storage condition also remained consistent over time, with sample pH remaining at pH 6.3 ± 0.1 over the course of three months.

[0252] Size exclusion chromatography (SEC) was used to assess the amount of aggregates and degradation products present in ENPP1 polypeptide formulation samples. The abundance of total high molecular weight (HMW) species, low molecular weight (LMW) species, and major peak purity over 12 months (5° C. and 25° C.) and 6 months (40° C.) are reported. [Table 19] [Table 20] [Table 21]

[0253] The specific activity and SEC data indicate that formulation C1, like formulations A and B (above), provides remarkable overall stability for ENPP1-Fc, particularly over a range of temperatures, for at least 12 months.

[0254] Example 11: Treatment with lyophilized ENPP1 polypeptide formulations ENPP1-Fc can be administered with any of the aforementioned formulations disclosed. In some embodiments, ENPP1-Fc is administered in a formulation comprising 20 mM citrate, 2 mM calcium chloride, 175 mM sucrose, 82 mM (D) mannitol, and 0.05% w / v polysorbate 20 at about pH 6.3. The lyophilized formulation is reconstituted in a suitable reconstitution buffer (or sterile water) prior to administration to a subject in need thereof.

[0255] ENPP1-Fc is administered at one of the following selected doses: 0.2 mg / kg, 0.6 mg / kg, and 1.8 mg / kg, subcutaneously (SC) at least once or twice every other month, at least once or twice a month, three times a month, or at least once or twice a week.

[0256] The first dose of ENPP1-Fc may be administered on day 1. On day 8 and thereafter, ENPP1-Fc is administered to the subject twice a week at the selected dose of ENPP1. The doses may be administered at approximately the same time on each administration day. Injection sites are alternated, with no site being within 2 inches of any previous injection site within the past two weeks.

[0257] The selected dose of ENPP1-Fc is one of 0.2 mg / kg, 0.6 mg / kg, or 1.8 mg / kg SC. The first dose of ENPP1-Fc may be administered on day 1. After the first dose, the subject may be observed for 7 days to monitor safety and collect PK samples. On day 8 and thereafter, the subject receives the selected dose twice weekly. Administration of ENPP1-Fc at the selected dose is continued if deemed appropriate by a medical professional.

[0258] Subjects may receive eight doses of ENPP1-Fc over the course of a 29 day period, for example, 0.2 mg / kg, 0.6 mg / kg, and 1.8 mg / kg doses resulting in exposures of 1.6 mg, 4.8 mg, and 14.4 mg every 29 days, respectively. Alternatively, subjects may receive as many as eight doses, as deemed appropriate by a medical professional.

[0259] Similar to the endogenous ENPP1 enzyme, ENPP1-Fc cleaves ATP to generate AMP and PPi, thereby increasing plasma PPi levels and increasing AMP which CD73 rapidly converts to adenosine. Supplementation of the endogenous human enzyme is intended to correct the underlying deficiency and allow for improved health and reduced clinical complications associated with ENPP1 baseline patient, clinician, and caregiver outcomes.

[0260] Example 12: Treatment of patients with ENPP1 deficiency Enpp1-Fc formulated as described above will be administered to patients identified as having an ENPP1 deficiency by subcutaneous injection twice weekly starting on days 1 and 8 using selected doses as follows: [Table 22]

[0261] ENPP1-Fc is administered at a selected dose of ENPP1-FC, one of 0.2 mg / kg, 0.6 mg / kg, or 1.8 mg / kg SC, at least twice weekly for a period of time determined by a medical professional. The patient's response to enzyme replacement is monitored as needed, e.g., following reduction in one or more symptoms of ENPP1 deficiency and / or as determined by a medical professional using the guidance provided herein.

[0262] Example 13: Treatment of Patients Diagnosed with GACI Enpp1 deficiency can be masked as GACI, a rare disease that occurs in infants and is associated with widespread arterial calcification (Albright, et al., 2015, Nature Comm. 10006).

[0263] ENPP1-Fc formulated as above is administered at a selected dose of ENPP1-FC, one of 0.2 mg / kg, 0.6 mg / kg, or 1.8 mg / kg SC, at least twice weekly for a period of time determined by a medical professional. The GACI patient's response is monitored as needed, e.g., following a reduction in one or more symptoms of GACI and / or as determined by a medical professional using the guidance provided herein.

[0264] Example 14: Treatment of patients diagnosed with ARHR2 Enpp1 deficiency can be masked as ARHR2, a rare skeletal disorder characterized by low levels of plasma PPi and serum phosphate, which can lead to rickets, repeated fractures of long bones, rachitic skeletal deformities, and impaired growth and development (Ferreira et al. 2014, Moran 1975, Rutsch et al. 2008).

[0265] ENPP1-Fc formulated as above is administered at a selected dose of ENPP1-Fc, one of 0.2 mg / kg, 0.6 mg / kg, or 1.8 mg / kg SC, at least twice weekly for a period of time determined by a medical professional. The response of the ARHR2 patient is monitored as needed, e.g., following a reduction in one or more symptoms of ARHR2 and / or as determined by a medical professional using the guidance provided herein.

[0266] The following examples provide guidance for determining treatment protocols and efficacy in the context of the entire specification.

[0267] Example 15: Measurement of inorganic pyrophosphate in plasma Low plasma PPi levels are a hallmark of ENPP1 deficiency and are used as an indicator of therapeutic efficacy. ENPP1-Fc specifically cleaves ATP to generate AMP and PPi. The therapeutic goal of ENPP1 ERT is to normalize extracellular PPi levels and correct clinical abnormalities associated with ENPP1 deficiency.

[0268] PPi is measured by obtaining a patient plasma sample. The determined PPi data may be used to adjust dose levels. PPi levels may also serve as a primary PD marker for PK / PD analysis.

[0269] The concentrations of Pi and PPi in mammals are 1 to 3 mM and 2 to 3 pM, respectively.

[0270] Example 16: Biomarkers associated with bone health In addition to low plasma PPi, patients with ENPP1 deficiency are biochemically characterized by low serum phosphate, high urinary phosphate, low renal TmP / GFR, normal calcium (Ca), low normal urinary Ca, normal 25-hydroxyvitamin D (25 OH D), low normal 1,25(OH)2D, high BAP, high intact FGF23, and normal PTH (IOF 2019).

[0271] Biomarkers that can be used as additional determinants of bone health in treated patients are shown in Table 19. [Table 23]

[0272] Example 17: Efficacy of treatment with ENPP1-Fc Treatment efficacy may be assessed by measuring plasma PPi and other plasma analytes such as FGF23, Pi, FGF23, Pi, TmP / GFR, serum alkaline phosphatase (ALP), bone-specific ALP (BALP), carboxy-terminal cross-linked telopeptide of collagen type I (CTx), and N-terminal propeptide of procollagen type 1 (P1NP). These analyte measurements can be used as PD markers associated with ENPP1 deficiency to determine the efficacy of ENPP1-FC. Changes in these analytes can be described as changes from baseline and in a time-dependent manner over the course of treatment. Dose linearity of PK and PD parameters may also be assessed.

[0273] Changes from baseline in plasma PPi levels, FGF23 levels, and urinary phosphate excretion per creatinine clearance may be analyzed using a paired difference t-test to test the null hypothesis that the change from baseline in PPi levels is equal to zero.

[0274] Example 18: Drug concentration measurement In addition, blood samples may be obtained from the patient for measurement of ENPP1-FC concentrations in plasma and for subsequent determination of PK parameters after the first dose (i.e., single dose) and after multiple doses (i.e., steady state) and thereafter.

[0275] Example 19: Immunogenicity (anti-drug antibodies) If desired, immunogenicity against ENPP1-Fc may be measured using anti-drug antibodies (ADA). Immunogenicity testing may utilize a multi-tiered approach, and if ADA is detected in the initial screening, confirmatory testing may be performed to determine specificity. Samples may also be used to assess and further establish specificity confirmation (i.e., titer) and neutralizing antibody assays.

[0276] Example 20: Pharmacokinetic, Pharmacodynamic, and Exploratory Biomarker Analysis Pharmacokinetic analysis may be performed on the PK population, and PK parameters of ENPP1-FC may be summarized by treatment with descriptive statistics. Dose linearity of PK and PD parameters may also be evaluated. PK / PD analysis, immunogenicity analysis, and exploratory biomarker analysis may be determined.

[0277] Example 21: Additional Determinants of Efficacy Restoration of normal levels of PPi is the primary indicator of the effectiveness of treatment with NEPP1-Fc, although other physical measurements can be used, if desired, to aid in determining the effectiveness of treatment. These include one or more of the following:

[0278] 1. X-ray and imaging X-rays of skeletal severity. Standard x-rays may be obtained to detect the skeletal deformities of rickets. Acquired x-rays may be taken, for example, on the wrists and knees.

[0279] DEXA scan. A DEXA scan may be used to assess changes in bone density.

[0280] Positron emission tomography-computed tomography. Baseline Na 18 F-PET / HRpQCT (or HR-CT) may be a whole-body scan performed within one month of the first dose of ENPP1-FC to measure arterial and organ calcification and skeletal abnormalities for baseline and future intervention assessment. 18 F-PET measures bone metabolism and arterial microcalcification. High-resolution quantitative computed tomography (HRQCT) or HR-CT can determine bone microarchitecture in the non-dominant distal extremity and tibia. Standard bone geometric parameters are calculated.

[0281] Doppler echocardiogram. A baseline echocardiogram may be obtained within 3 days prior to the first dose of ENPP1-FC. Doppler echocardiogram can be used to measure cardiac and valvular cardiac function [LVEF, blood flow] calcification, and arteriosclerosis.

[0282] Optical coherence tomography. Optical coherence tomography may be used to visualize neointimal proliferation.

[0283] Peripheral Arterial Tonometry. Peripheral arterial tonometry (PAT) may be used to assess digital pulse wave amplitude (PWA), which corresponds to digital stroke volume variation.

[0284] Renal Ultrasound. Renal ultrasound may be used to measure kidney calcification, for example, within one week of initiating ENPP1-FC.

[0285] Bone histomorphology and bone biopsy. A bone biopsy may be performed as a baseline measurement. A 10 day tetracycline challenge prior to bone biopsy is preferred.

[0286] 2. Walking ability. Walking tests may be used as submaximal exercise measures to measure functional capacity in ambulatory patients combining cardiopulmonary, neuromuscular, and musculoskeletal function. The 6-minute walk test (6MWT) was originally developed by the American Thoracic Society (ATS 2002) for use in adults and is now commonly used in both adult and pediatric populations (Mylius et al. 2016), as well as in children with neuromuscular diseases such as spinal muscular atrophy (Montes et al. 2018), Duchenne muscular dystrophy (McDonald et al. 2013), and infantile-onset Pompe disease (van der Meijden et al. 2018). The 2-minute walk test (2MWT) is included in the NIH toolbox and is increasingly used to measure the same characteristics.

[0287] The 6MWT and 2MWT may be administered to patients before and during treatment at the discretion of the healthcare provider. If the subject is unable to complete at least the 2MWT at baseline, additional assessments during treatment may be at the discretion of the healthcare provider. Resting heart rate is obtained before and after the test. Distance walked during the first 2 minutes and the entire 6 minutes of the 6MWT may be recorded. Distance walked during the 2 and 6 minutes may be compared to age- and sex-matched normative data (percent predicted).

[0288] 3. Dynamometry. Strength may be assessed using dynamometry prior to and / or during treatment at the discretion of the healthcare provider. Handheld dynamometry is a direct measurement of strength commonly used in both children and adults. Muscle groups that may be assessed include shoulder abduction, shoulder flexion, elbow flexion, elbow extension, hip abduction, hip flexion, hip extension, and knee extension. Each muscle group may be measured twice on both sides.

[0289] Grip Strength. Grip strength may be measured using a grip strength dynamometer prior to and / or during treatment at the discretion of the healthcare provider. Equipment and assessor instructions may be standardized across centers. Grip strength may be assessed bilaterally with one exercise and one maximum force measurement taken for each hand, and results may be compared to age- and sex-matched normative data (if available).

[0290] Range of Motion. Range of motion may be assessed using a goniometer, an instrument that tests joint angles and measures the degree of motion at a joint. The fixed arm of the goniometer aligns with a designated bony landmark on a fixed body segment, and the moving arm of the goniometer aligns with a designated bony landmark on the moving limb. A fulcrum of the goniometer is designated for each motion measured using the axis of motion and the bony landmark. Range of motion may be assessed for one or more of shoulder abduction, shoulder flexion, elbow flexion, elbow extension, hip abduction, hip flexion, hip extension, and knee extension.

[0291] 4. Hearing Testing. Moderate hearing loss is associated with ARHR2 (Brachet et al 2014, Steichen-Gersdorf et al 2015). Baseline hearing may be determined by one or more of the following: physical examination and otoscopy, impedance hearing test (commonly referred to as tympanometry), pure tone audiometry (PTA) (frequencies up to 8 kHz, if available). (If PTA thresholds are >15 dB, subjects should also undergo bone conduction testing), high frequency audiometry (HFA) (frequencies up to 16 kHz).

[0292] 5. Clinician Global Impression Scale. The Clinical Global Impression (CGI-S) scale was developed for use in clinical trials sponsored by the National Institute of Mental Health to provide a brief, independent assessment of the clinician's perspective of a patient's overall functioning before and after starting an investigational drug (Guy 1976). The CGI provides a clinician-determined overall summary measure that considers all available information, including knowledge of the patient's medical history, psychosocial situation, symptoms, behavior, and the impact of symptoms on the patient's ability to function. The CGI-S may be administered before and / or during treatment at the discretion of the healthcare provider and provides a global assessment of change using a 7-point scale ranging from -3 (severe worsening) to +3 (marked improvement).

[0293] 6. Gross Motor Function Classification System-Extended and Revised. The Gross Motor Function Classification System-Extended and Revised (GMFCS-E and R) may be administered prior to and / or during treatment at the discretion of the healthcare provider. GMFCS-E and R classify patient-initiated movements with an emphasis on mobility on a scale of 1 to 5.

[0294] 7. Patient-Reported Outcomes Measurement Information System. The Patient-Reported Outcomes Measurement Information System (PROMIS) consists of a variety of questionnaires developed by the US National Institutes of Health (NIH) to assess physical, mental, and social health from the patient's perspective (http: / / www.healthmeasures.net). These questionnaires have been used in clinical trials in people with chronic health conditions such as X-linked hypophosphatemia, arthritis, multiple sclerosis, and neurofibromatosis. Each questionnaire contains 8–10 items that participants rate on a 5-point Likert scale ranging from 1 (never) to 5 (always). The scores of each questionnaire are summed, with higher scores indicating more measured domains (e.g., higher fatigue, higher physical function). Raw scores are converted to T-scores based on a mean of 50 and a standard deviation of 10, allowing comparison of the study sample to the general population. PROMIS scales may include Pain Interference (Short Form 8a), Pain Intensity (Version 3a), Physical Function-Upper Extremity (Custom Short Form), Physical Function-Mobility (Short Form 13a FACIT Fatigue), Fatigue (Short Form), and Cognitive Impact (Short Form 8a) and may be administered before and / or during treatment at the discretion of the healthcare provider. These assessments may be completed by the subject unassisted.

[0295] 8. Caregiver Global Impression Scale. The Caregiver Global Impression of Status may be administered to the patient's caregiver prior to and / or during treatment, at the discretion of the healthcare provider. The Caregiver Global Impression of Change provides an overall assessment of change using a 7-point scale ranging from -3 (severe deterioration) to +3 (marked improvement).

[0296] 9. Western Ontario-McMaster Universities Osteoarthritis Index. WOMAC is a patient-reported outcome used to assess activities of daily living, functional mobility, gait, general health, pain, and quality of life in patients with low back or knee pain (www.sralab.org). The assessment consists of 24 items and takes approximately 12 minutes to administer. WOMAC may be administered before and / or during treatment at the discretion of the healthcare provider. The assessment can be completed unassisted by the subject.

[0297] Example 22: Long-term stability study of lyophilized ENPP1 polypeptide formulations Lyophilized ENPP1-Fc in the preferred formulation was tested for long-term stability when stored at three different storage conditions, 5°C, 25°C, and 40°C, over a period of 36 months. After 24 months, all lyophilized cakes containing ENPP1-Fc showed an intact structure with an ivory color. After reconstitution in water, the appearance of the liquid was observed to be brownish yellow, clear, and free of visible particles for all sample types. Reconstitution occurred within a window consistent with the data described above, with complete reconstitution of the lyophilized samples occurring within 59 to 108 seconds.

[0298] The moisture content of both formulations over temperature conditions over time also remained consistent with the moisture content data described above. All lyophilized sample time points (0, 3, 6, 9, 12, 18, 24, 30, 36 months) at all of the storage temperatures tested had very low or no moisture content across time points and conditions, with residual moisture content ranging from 0.0% to 0.3%. The pH value of each sample at each storage condition also remained consistent over time, with sample pH remaining at pH 6.3 ± 0.1 over the course of 36 months. Protein recovery relative to a normalized starting concentration of 50 mg / mL was also measured for each sample. These data indicate that protein recovery does not degrade over time under storage conditions at 5° C., 25° C., and 40° C.

[0299] SEC experiments were performed to characterize the tendency of samples of the preferred formulations to develop HMW species over a 36 month period when stored at 5°C, 25°C, and 40°C. Summary SEC data for total HMW, LMW, and major peak abundance were collected following the procedures outlined in the previous examples. Figures 17-19 show summary SEC data for HMW abundance and major peak abundance over time at 5°C, 25°C, and 40°C, respectively. The positive control zero time point sample had an HMW content of approximately 4%. All preferred formulation samples remained consistent with the control, with ±5% HMW content over a period of at least 24 months under storage conditions of 5°C, 25°C, and 40°C, respectively. These results indicate that each preferred formulation is surprisingly capable of preventing the formation of high molecular weight aggregate species over an extended period of time. Similar results were seen for samples stored for up to 36 months. These data also show that preferred formulations are able to stabilize lyophilized polypeptides against the formation of HMW species even when stored at higher or non-refrigerated temperatures, such as 25° C. and 40° C.

[0300] The specific activity for all preferred formulation samples corresponding to each time point and storage condition was evaluated using the enzyme activity screen described above in Example 7. Briefly, the enzyme assay used to evaluate the activity of each formulation sample was performed by mixing ENPP1-Fc with pNP-TMP. Product formation (pNP) is monitored spectrophotometrically at 405 nm for a period of 5 minutes at 25°C. The specific activity of ENPP1-Fc is calculated by interpolating the change in absorbance over time obtained for each sample preparation to a standard curve of pNP of known concentration. Activity data was plotted over time for each sample, including the frozen control. Representative activity versus time plots for samples stored at 5°C, 25°C, and 40°C for up to 24 months are shown in Figure 20. Similar results were seen for samples stored for up to 36 months.

[0301] All samples across time points and storage conditions showed consistent levels of enzyme activity, thus demonstrating that the lyophilized ENPP1-Fc formulation remains stable under long-term storage even when stored under different temperature conditions.

[0302] Example 23: Stability analysis of reconstituted samples in vials and syringes The following assays were performed to determine whether the reconstituted formulations suffered any protein loss or loss of activity over time when stored in vials and syringes at different time points.

[0303] Lyophilized ENPP1-Fc in the preferred formulation was reconstituted in water to achieve different concentrations ranging from 1 mg / ml, 2.5 mg / ml, and 10 mg / ml. Samples of different concentrations were incubated at 5° C. in two sets of syringes (high volume sample-250 μl and low volume sample-50 μl) for different time intervals ranging from 1 to 12 hours. The experiment was repeated three times. Samples reconstituted at the time of assay and diluted to 10, 2.5, and 1 mg / mL were used as controls. The enzyme activity of the unincubated samples (time-0 hours) was determined. The activity of the incubated samples (8-12 hours) was then normalized to the control value. Protein recovery relative to a normalized starting concentration of 50 mg / mL was also measured for each sample following the protocol described above in the preceding examples.

[0304] FIG. 21 shows the recovery of reconstituted ENPP1-Fc stored in syringes or vials for different time intervals. These data indicate that reconstituted ENPP1-Fc protein is stable when stored in syringes at different concentrations. The specific activity of reconstituted ENPP1-Fc samples was measured according to the protocol described above in the previous examples. FIG. 22 shows a summary of enzyme activity data for reconstituted ENPP1-Fc polypeptide samples stored in syringes or vials for different time intervals. All samples across time points and storage conditions showed consistent levels of enzyme activity. Protein activity did not change when stored in vials, but there was a slight loss of activity when samples were stored in syringes for more than 8 hours.

[0305] Example 24: Stability analysis of reconstituted samples after dilution Lyophilized ENPP1-Fc formulations in vials are reconstituted to 25 mg / mL. Two sets of dilutions are made at 2.5 mg / mL and 1 mg / mL. Control samples were made by taking separate vials and diluting to 2.5 mg / mL and 1 mg / mL at the time of the assay using a syringe. Thus, the control sample is at zero incubation time. The diluted samples 2.5 mg / mL and 1 mg / mL were incubated overnight at 5° C., and a subset of samples was incubated at room temperature (25° C.) for time intervals of 1 hour, 2 hours, 3 hours, 4 hours, and 8 hours. Enzyme activity and protein concentration were measured as described above in the previous examples. Values ​​were normalized to the control sample. Figure 23 shows the recovery of reconstituted ENPP1-Fc stored in syringes or vials at different time intervals. The assay showed that there was no loss in protein concentration when stored overnight in vials at 5° C. or at room temperature for up to 8 hours. Figure 24 shows a summary of enzyme activity data for reconstituted ENPP1-Fc polypeptide samples stored in syringes or vials for different time intervals. The assay showed no loss in protein activity when stored overnight in vials at 5°C or at room temperature for up to 4 hours. A small loss of enzyme activity was observed when samples were stored at room temperature after 4 hours.

[0306] References ATS. American Thoracic Society Statement: Guidelines for the six - minute walk test. Am J Respir, Crit Care Med. 2002 2002;166(1):111 - 7. Brachet C, Mansbach AL, Clerckx A, Deltenre P, Heinrichs C. Hearing loss is part of the clinical picture of ENPP1 loss of function mutation. Horm Res Paediatr. 2014;81(1):63 - 6. CTFG. Recommendations related to contraception and pregnancy testing in clinical trials. 2014 [May 19, 2020]; Available from: http: / / www.hma.eu / fileadmin / dateien / Human_Medicines / 01 - About HMA / Working Groups / CTFG / 2014 09 HMA CTFG Contraception.pdf. Ferreira C, Ziegler S, Gahl W. Generalized Arterial Calcification of Infancy. In: Adam MP, Ardinger HH, Pagon RA, Wallace SE, Bean LJH, Stephens K, et al., editors. GeneReviews((R)). Seattle(WA)2014. Guy W. The Clinical Global Impression Scale. In: Rush Jr AJ, First MB, Blacker D, editors. Handbook of Psychiatric Measures, 2008. Washington, DC: American Psychiatric Publishing, Inc; 1976. p. 90-2. IOF. Autosomal Recessive Hypophosphatemic Rickets Type 2 (ARHR2). 2019 [May 19, 2020]; Available from: https: / / www.iofbonehealth.org / osteoporosis-musculoskeletal-disorders / skeletal-rare-disorders / autosomal-recessive-hypophosphatemi-0. Mackenzie NC, Huesa C, Rutsch F, MacRae VE. New insights into NPP1 function: lessons from clinical and animal studies. Bone. 2012 Nov;51(5):961-8. McDonald CM, Henricson EK, Abresch RT, Florence JM, Eagle M, Gappmaier E, et al. The 6-minute walk test and other endpoints in Duchenne muscular dystrophy: longitudinal natural history observations over 48 weeks from a multicenter study. Muscle Nerve. 2013 Sep;48(3):343-56. Montes J, McDermott MP, Mirek E, Mazzone ES, Main M, Glanzman AM, et al. 脊髓性肌萎缩症的行走功能:与年龄相关的进展模式。《公共科学图书馆·综合》。2018年;13(6):e0199657。 莫兰·J·J。婴儿特发性动脉钙化:一项临床病理研究。《病理学年鉴》。1975年;10:393 - 417。 米利乌斯·C·F,帕普·D,塔肯·T。儿童和青少年6分钟步行试验的参考值:一项系统评价。《专家评论:呼吸医学》。2016年12月;10(12):1335 - 52。 美国国立癌症研究所。美国国立癌症研究所癌症治疗与诊断司(DCTD),美国国立癌症研究所(NCI),美国国立卫生研究院(NIH),卫生与公众服务部(DHHS)。不良事件通用术语标准第5.0版(CTCAE)。发布日期:2017年11月27日。可在以下网址获取https: / / ctep.cancer.gov / protocoldevelopment / electronic_applications / docs / ctcae_v5_quick_re ference 8.5x11.pdf。2017年。 奥里斯·I·R,阿内特·T·R,拉塞尔·R·G。焦磷酸盐:矿化的关键抑制剂。《当代药理学观点》。2016年6月;28:57 - 68。 Rutsch F, Boyer P, Nitschke Y, Ruf N, Lorenz-Depierieux B, Wittkampf T, et al.Hypophosphatemia, hyperphosphaturia, and bisphosphonate treatment are associated with survival beyond infancy in generalized arterial calcification of infancy.Circ Cardiovasc Genet.2008 Dec;1(2):133-40. Steichen-Gersdorf E, Lorenz-Depiereux B, Strom TM, Shaw NJ.Early onset hearing loss in autosomal recessive hypophosphatemic rickets caused by loss of function mutation in ENPP1.J Pediatr Endocrinol Metab.2015 Jul;28(7-8):967-70. van der Meijden JC, Kruijshaar ME, Harlaar L, Rizopoulos D, van der Beek N, van der Ploeg AT.Long-term follow-up of 17 patients with childhood Pompe disease treated with enzyme replacement therapy.J Inherit Metab Dis.2018 Nov;41(6):1205-14.

[0307] Incorporation by Reference All publications and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0308] While specific embodiments of the subject matter have been discussed, the above specification is illustrative and not limiting. Many variations will become apparent to those skilled in the art upon review of this specification and the following claims. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

1. A lyophilized polypeptide formulation comprising: (a) an ENPP1 polypeptide; and (b) one or more of a buffer, a stabilizer, a salt, and a surfactant.

2. the lyophilized polypeptide formulation comprises a buffer; Optionally, the buffering agent is: (i) when reconstituted in a solution, it maintains a pH range of pH 6-7 or pH 7-8, preferably the buffer is selected from the group consisting of succinate, citrate, bicarbonate, phosphate, Tris, or glycylglycine, particularly preferably the buffer is succinate, citrate, or phosphate; (ii) the buffering agent, when reconstituted in solution, has a concentration ranging from 5 mM to 100 mM; (iii) the buffering agent increases the onset temperature of aggregate formation; (iv) the buffering agent increases the onset temperature of aggregate formation by at least 2°C; (v) the buffer reduces the formation of high molecular weight species, and / or (vi) The formulation of claim 1, wherein the buffering agent reduces the formation of high molecular weight species by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%.

3. the lyophilized polypeptide formulation comprises one or more pharmaceutically acceptable excipients; Optionally, said one or more pharmaceutically acceptable excipients (i) a stabilizer, an amino acid, a salt, a metal ion, and a surfactant, preferably wherein the stabilizer is a sugar, a carbohydrate, or a polysaccharide, more preferably wherein the sugar is selected from the group consisting of sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, or sorbitol; (ii) mannitol, sucrose, or an amino acid, optionally wherein the amino acid is selected from the group consisting of glycine, arginine, histidine, alanine, proline, serine, and glutamic acid; (iii) arginine; (iv) a salt, optionally selected from the group consisting of sodium chloride (NaCl), calcium chloride (CaCl2), zinc chloride (ZnCl2), and / or magnesium chloride (MgCl2); (v) calcium chloride (CaCl 2 ); (vi) a surfactant, optionally the surfactant being selected from the group consisting of polysorbate, poloxamer, triton, sodium dodecyl sulfate, sodium laurel sulfate, sodium octyl glycoside, lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, stearyl sulfobetaine, lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, stearyl sarcosine, linoleyl betaine, myristyl betaine, cetyl betaine, lauroamidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmidopropyl betaine, isostearamidopropyl betaine, myristamidopropyl dimethylamine, palmidopropyl dimethylamine, isostearamidopropyl 2. The formulation of claim 1, wherein the surfactant is selected from the group consisting of: pyr-dimethylamine, sodium methyl cocoyl taurate, disodium methyl oleate, dihydroxypropyl PEG-5 linoleate ammonium chloride, polyethylene glycol, polypropylene glycol, polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, polysorbate 188, PEG 3350, and mixtures thereof; more preferably, the surfactant is selected from the group consisting of polysorbate 20 (PS20), polysorbate 80 (PS80), or polysorbate 188 (PS188); particularly preferably, the surfactant is polysorbate 20 (PS20).

4. The lyophilized polypeptide formulation comprises a buffer, a stabilizer, a salt, an amino acid, and a surfactant, and optionally (i) the buffering agent is citrate or succinate, the stabilizing agent is sucrose or mannitol, the salt is calcium chloride (CaCl 2 ), the surfactant is PS20, and optionally the formulation comprises 75 mM to 95 mM each of sucrose, mannitol, or a combination thereof; (ii) the pH of the formulation is between pH 6.2 and pH 6.5, or the formulation comprises 15 to 25 mM citrate; optionally, the formulation comprises 75 mM to 95 mM each of sucrose, mannitol, or a combination thereof; (iii) The formulation of claim 1, wherein the formulation comprises 100 mM to 300 mM sucrose, 1 mM to 3 mM calcium chloride, and / or 0.005% to 0.1% (w / v) PS20.

5. the one or more pharmaceutically acceptable excipients are present at a concentration in the range of 5 mM to 300 mM when reconstituted in solution; 2. The formulation of claim 1, wherein preferably the one or more pharmaceutically acceptable excipients reduce the formation of high molecular weight species, more preferably said reduction is at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%.

6. The lyophilized polypeptide formulation comprises an ENPP1 polypeptide cofactor, and optionally: (i) the ENPP1 polypeptide cofactor comprises calcium, zinc, and / or adenosine monophosphate; (ii) the ENPP1 polypeptide cofactor is CaCl2, CaSO4, ZnCl2, ZnSO4, and / or adenosine monophosphate, preferably the ENPP1 polypeptide cofactor is CaCl2 and / or adenosine monophosphate; (iii) the ENPP1 polypeptide cofactor, when reconstituted in solution, is present at a concentration ranging from 1 mM to 10 mM, or the ENPP1 polypeptide cofactor reduces the formation of high molecular weight species by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. (iv) the formulation comprises a surfactant, preferably the surfactant comprises PS20, PS80 or PS188, more preferably the surfactant comprises PS20, and / or the surfactant is present at a concentration in the range of 0.02% to 0.10% (w / v) when reconstituted in solution, and / or the surfactant increases resistance to physical stress; (v) the formulation has a uniform particle size; (vi) the formulation contains a 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold reduction in larger sized particles as determined by microflow imaging analysis; (vii) the ENPP1 polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence comprising SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15, and / or the ENPP1 polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence comprising SEQ ID NO:2; (viii) the ENPP1 polypeptide is a fusion protein comprising a soluble ENPP1 polypeptide domain and one or more heterologous protein moieties, preferably wherein the heterologous protein moieties increase the circulating half-life of the soluble ENPP1 polypeptide in a mammal, more preferably wherein the heterologous protein moieties comprise an Fc domain, and even more preferably wherein the Fc domain comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 12, and particularly preferably wherein the ENPP1 polypeptide is an ENPP1-Fc fusion protein; and / or (ix) The formulation of claim 1, wherein the ENPP1 polypeptide further comprises a heterologous moiety, optionally wherein the heterologous moiety is selected from the group consisting of a glycosylated amino acid, a PEGylated amino acid, a farnesylated amino acid, an acetylated amino acid, a biotinylated amino acid, and a lipid moiety.

7. 2. The formulation of claim 1, wherein the ENPP1 polypeptide comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to an amino acid sequence comprising SEQ ID NO:13, SEQ ID NO:14, and / or SEQ ID NO:

15.

8. The lyophilized polypeptide formulation comprises an ENPP1 polypeptide, a buffer, a pharmaceutically acceptable salt, a stabilizer, a surfactant, and one or more pharmaceutically acceptable excipients, and optionally: (i) the buffer is a citrate buffer, more preferably the citrate buffer is trisodium citrate dihydrate; (ii) the pharmaceutically acceptable salt is calcium chloride; (iii) the stabilizing agent is a sugar, more preferably the stabilizing agent is sucrose; and / or (iv) The formulation of claim 1, wherein the one or more pharmaceutically acceptable excipients comprises mannitol. (i) the lyophilized polypeptide formulation comprises an ENPP1 polypeptide, a citrate buffer, calcium chloride, sucrose, and polysorbate; (ii) the lyophilized polypeptide formulation comprises an ENPP1 polypeptide, trisodium citrate dihydrate, calcium chloride dihydrate, sucrose, and polysorbate 20; (iii) the lyophilized polypeptide formulation comprises an ENPP1 polypeptide, a citrate buffer, calcium chloride, sucrose, mannitol, and polysorbate; (iv) the lyophilized polypeptide formulation comprises an ENPP1 polypeptide, trisodium citrate dihydrate, calcium chloride dihydrate, sucrose, D(-)mannitol, and polysorbate 20; (v) the lyophilized polypeptide formulation comprises, per 0.5 ml final reconstitution volume, about 25 mg of ENPP1 polypeptide, about 2.94 mg of trisodium citrate dihydrate, about 0.15 mg of calcium chloride dihydrate, about 30 mg of sucrose, about 7.5 mg of D(-)mannitol, and about 0.25 mg of polysorbate; (vi) the ENPP1 polypeptide comprises or consists of SEQ ID NO: 13, 14 or 15; (vii) the ENPP1 polypeptide further comprises an Fc domain, and optionally the Fc domain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 12, and / or the ENPP1 polypeptide comprises a linker amino acid sequence joining the ENPP1 polypeptide moiety and the Fc domain; (viii) the linker amino acid sequence present in the ENPP1 polypeptide present in said lyophilized polypeptide formulation comprises or consists of the amino acid sequence LIN; and / or (ix) The formulation of claim 1, wherein the amino acid sequence of the ENPP1 polypeptide present in the lyophilized polypeptide formulation consists of or comprises SEQ ID NO: 9, SEQ ID NO: 10, or SEQ ID NO:

11.

10. 10. The formulation of any one of claims 1 to 9, wherein the lyophilized polypeptide formulation is reconstituted in a sterile injection solution, optionally comprising: (i) the lyophilized polypeptide formulation is reconstituted in a reconstitution solution or sterile water, preferably the reconstitution solution comprises a pharmaceutically acceptable carrier and / or excipient, more preferably the pharmaceutically acceptable carrier is selected from saline, purified water, or sterile water for injection; (ii) the lyophilized polypeptide formulation is completely reconstituted within a period of less than 100 seconds, 80 seconds, 70 seconds, 68 seconds, 65 seconds, or 60 seconds; and / or (iii) the reconstituted formulation comprises at least or about 50 mg / mL of the ENPP1 polypeptide, about 20 mM citrate at about pH 6.3, about 2 mM calcium chloride, about 175 mM sucrose, about 82 mM mannitol, and about 0.05% w / v polysorbate, or the reconstituted formulation comprises at least or about 50 mg / mL of the ENPP1 polypeptide, about 20 mM citrate at about pH 6.3, about 2 mM calcium chloride, about 175 mM sucrose, about 82 mM (D) mannitol, and about 0.05% w / v polysorbate. a formulation comprising at least or about 50 mg / mL of the ENPP1 polypeptide, about 20 mM citrate at about pH 6.3, about 88 mM sucrose, about 82 mM mannitol, about 2 mM calcium chloride, and about 0.05% polysorbate 20, or a formulation comprising at least or about 50 mg / mL of the ENPP1 polypeptide, about 20 mM citrate at about pH 6.3, about 263 mM sucrose, about 2 mM calcium chloride, and about 0.05% polysorbate 20.

11. 11. The formulation of claim 10, wherein the reconstituted formulation exhibits long-term stability at -80°C to 40°C; optionally, the formulation has a shelf life of at least 3, 6, 12, 24, 36, 48, or 60 months.

12. 11. The formulation of claim 10, wherein the reconstituted formulation has a shelf life of at least 1, 2, 3, 4, 5, 6, 12, 18, 24, 48, or 60 hours.

13. 10. A method for producing a pharmaceutical solution comprising an ENPP1 polypeptide, comprising contacting a lyophilized polypeptide formulation of any one of claims 1 to 9 with a sterile reconstitution solution or sterile water, thereby producing a reconstituted solution comprising said ENPP1 polypeptide, and optionally (i) the reconstituted solution comprises a pharmaceutically acceptable carrier and / or excipient, optionally wherein the pharmaceutically acceptable carrier is selected from saline, purified water, or sterile water for injection; (ii) the lyophilized polypeptide formulation is completely reconstituted within a period of less than 100 seconds, 80 seconds, 70 seconds, 68 seconds, 65 seconds, or 60 seconds; and / or (iii) the reconstituted formulation comprises at least or about 50 mg / mL of the ENPP1 polypeptide, about 20 mM citrate at about pH 6.3, about 2 mM calcium chloride, about 175 mM sucrose, about 82 mM mannitol, and about 0.05% w / v polysorbate; the reconstituted formulation comprises at least or about 50 mg / mL of the ENPP1 polypeptide, about 20 mM citrate at about pH 6.3, about 2 mM calcium chloride, about 175 mM sucrose, about 82 mM (D) mannitol, and about 0.05% w / v polysorbate 20; the reconstituted formulation comprises at least or about 50 mg / mL of the ENPP1 polypeptide, about 20 mM citrate at about pH 6.3, about 88 mM sucrose, about 82 mM mannitol, about 2 mM calcium chloride, and about 0.05% polysorbate 20; or The method, wherein the reconstituted formulation comprises at least or about 50 mg / mL of the ENPP1 polypeptide, about 20 mM citrate at about pH 6.3, about 263 mM sucrose, about 2 mM calcium chloride, and about 0.05% polysorbate 20.

14. A reconstituted solution produced by the method of claim 13.

15. A formulation comprising a therapeutically effective dose of a lyophilized polypeptide formulation according to any one of claims 1 to 9, the formulation optionally comprising reconstituted lyophilized ENPP1 polypeptide.

16. A vial containing the formulation of claim 15, the vial optionally containing reconstituted lyophilized ENPP1 polypeptide.

17. A syringe comprising the formulation of claim 15, optionally containing reconstituted lyophilized ENPP1 polypeptide.

18. (i) For use in preventing the progression of or reducing vascular calcification in a subject in need thereof. (ii) for use in preventing the progression of or reducing pathological calcification in a subject with ENPP1 deficiency; (iii) For use in preventing the progression of or reducing tissue calcification in a subject in need thereof; (iv) for use in preventing the progression of pathological ossification or reducing pathological ossification in a subject with ENPP1 deficiency; (v) for use in increasing circulating pyrophosphate (PPi) in a subject in need thereof; or (vi) The formulation of claim 15 for use in increasing pyrophosphatase activity in a subject in need thereof.

19. The formulation of claim 18, wherein the subject has ENPP1 deficiency, a disorder associated with pathological calcification, a disorder associated with pathological ossification, or ABCC6 deficiency, and optionally, the ABCC6 deficiency is pseudoxanthoma elasticum (PXE).

20. 19. The formulation of claim 18, wherein the subject has or is at risk of developing pathologic soft tissue calcification, arterial calcification, vascular calcification, chronic kidney disease (CKD), end-stage renal disease (ESRD), uremic arteriolar calcification (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), or hypophosphatemic rickets. (i) for use in ameliorating one or more symptoms of ENPP1 deficiency in a subject. (ii) for use in treating a subject having an ENPP1 deficiency; (iii) for use in ameliorating one or more symptoms of ABCC6 deficiency in a subject; (iv) The formulation of claim 18 for use in treating a subject having an ABCC6 deficiency.

22. The formulation is a reconstituted formulation; (i) the reconstituted formulation is administered parenterally, via subcutaneous injection, via intravenous injection, via intradermal injection, or via intramuscular injection; (ii) the reconstituted formulation is self-administered, and / or (iii) The formulation of claim 18, wherein the formulation is administered several times daily, every two days, every three days, once a week, or once a month, and optionally, a second dose of the formulation is administered after a suitable time interval of at least 2 days, 4 days, 1 week, or 1 month.

23. The formulation of claim 18, wherein the formulation is a reconstituted formulation and comprises at least or about 50 mg / mL of the ENPP1 polypeptide, about 20 mM citrate at about pH 6.3, about 2 mM calcium chloride, about 175 mM sucrose, about 82 mM mannitol, and about 0.05% w / v polysorbate.

24. The formulation of claim 18, wherein the formulation is a reconstituted formulation and comprises at least or about 50 mg / mL of the ENPP1 polypeptide, about 20 mM citrate at about pH 6.3, about 2 mM calcium chloride, about 175 mM sucrose, about 82 mM (D) mannitol, and about 0.05% w / v polysorbate 20.

25. The formulation of claim 18, wherein the formulation is a reconstituted formulation and comprises at least or about 50 mg / mL of the ENPP1 polypeptide, about 20 mM citrate at about pH 6.3, about 88 mM sucrose, about 82 mM mannitol, about 2 mM calcium chloride, and about 0.05% polysorbate 20.

26. The formulation of claim 18, wherein the formulation is a reconstituted formulation and comprises at least or about 50 mg / mL of the ENPP1 polypeptide, about 20 mM citrate at about pH 6.3, about 263 mM sucrose, about 2 mM calcium chloride, and about 0.05% polysorbate 20.

27. The formulation of claim 18, wherein the formulation has a pH of 6.3 and is a reconstituted formulation comprising 25 mg / mL of the ENPP1 polypeptide, 20 mM citrate, 175 mM sucrose, 82 mM mannitol, 2 mM calcium chloride, and about 0.05% w / v polysorbate.

28. The formulation of claim 18, wherein the formulation has a pH of 6.3 and is a reconstituted formulation comprising approximately 10 mg / mL of the ENPP1 polypeptide, 8 mM citrate, 70.1 mM sucrose, 32.9 mM mannitol, 0.8 mM calcium chloride, and 0.02% w / v polysorbate.

29. (i) In the manufacture of a pharmaceutical composition for use in treating or preventing the progression of vascular calcification in a subject in need thereof, (ii) in the manufacture of a pharmaceutical composition for use in preventing the progression of or reducing pathological calcification in a subject with ENPP1 deficiency, (iii) in the manufacture of a pharmaceutical composition for use in preventing the progression of or reducing tissue calcification in a subject in need thereof, (iv) in the manufacture of a pharmaceutical composition for use in increasing circulating pyrophosphate (PPi) in a subject in need thereof; or (v) Use of a formulation according to any one of claims 1 to 9 in the manufacture of a pharmaceutical composition for use in increasing pyrophosphatase activity in a subject in need thereof.

30. The use of claim 29, wherein the subject has ENPP1 deficiency, a disorder associated with pathological calcification, a disorder associated with pathological ossification, or ABCC6 deficiency, and optionally the ABCC6 deficiency is pseudoxanthoma elasticum (PXE); or the subject has or is at risk of developing pathological soft tissue calcification, arterial calcification, vascular calcification, chronic kidney disease (CKD), end-stage renal disease (ESRD), uremic arteriolar calcification (CUA), calciphylaxis, ossification of the posterior longitudinal ligament (OPLL), or hypophosphatemic rickets.

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