Parathyroid hormone variants

Abstract

The present invention relates to parathyroid hormone (PTH) variants and pharmaceutical compositions comprising the same. The present invention further relates to PTH compositions having improved serum half-life and peak-to-trough ratio and methods for controlling serum calcium levels using the PTH variants and compositions of the present invention. The present invention further relates to methods for treating hypoparathyroidism and / or hypocalcemia caused by hypoparathyroidism using the PTH variants and compositions of the present invention.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 62 / 798,113 filed on January 29, 2019, U.S. Provisional Application No. 62 / 800,744 filed on February 4, 2019, and U.S. Provisional Application No. 62 / 884,730 filed on August 9, 2019, the disclosures of which are incorporated herein by reference in their entireties. Technical Field

[0003] The present invention relates to parathyroid hormone variants and compositions comprising the same. The present invention further relates to parathyroid hormone variants and compositions having improved serum half-life, and methods of controlling serum calcium levels using the parathyroid hormone compositions of the present invention. Background Art

[0004] Parathyroid hormone (PTH) is an 84-amino acid product secreted by the parathyroid glands of mammals that controls serum calcium levels through its effects on various tissues, including bone. Studies in humans with certain forms of PTH have shown that it has anabolic effects on bone and have generated great interest in its use in the treatment of osteoporosis and related bone diseases, as well as hypoparathyroidism.

[0005] The first 34 N-terminal amino acids are known to have the same activation effect as full-length PTH (1-84) on the only known receptor for PTH, "PTH1R". See, for example, Potts et al., Amer. Journ. of Med., 50:639-649 (1971); Potts et al., Proceed. of the 3rd Intern. Symp. of Endocrin., London, pp. 333-349 (1971); Tregear et al., Endocrin., 93:1349-1353 (1973). In addition, it has been shown that C-terminal fragments, such as 39-84 and 53-84, do not compete with the 1-34 fragment for receptor binding. In addition, these C-terminal fragments do not activate adenylate cyclase, all of which leads to the conclusion that the C-terminal portion of the PTH peptide is insignificant. See, Segre et al., J. of Bio. Chem., 254:6980-6986 (1979); Nissenson et al., J. of Bio. Chem., 254:1469-1475 (1979); Potts et al., Adv. in Prot. Chem., 35:323-396 (1982).

[0006] However, even though the C-terminus of PTH was thought to be irrelevant for biological activity, it has been found that the C-terminal portion of the peptide is required for normal trafficking and processing. See, e.g., Kemper et al., Proceed. of the Nat. Acad. of Sciences, 71:3731-3735 (1974); Freeman et al., Molec. Endocrin., 1:628-638 (1987); Wiren et al., Journal of Biochemistry, 263:19771-19777 (1988); Cioffi et al., Journal of Biochemistry, 264:15052-15058 (1989); Karaplis et al., Journal of Biochemistry, 270:1629-1635 (1995); Lim et al., Endocrinology, 131:2325-2330 (1992). In particular, there is evidence that full-length PTH is cleaved into C-terminal fragments within the parathyroid glands and that these fragments are secreted in response to increased calcium ion concentrations in the blood. To date, there is no evidence that the N-terminal fragment is stored within or secreted from the glands other than in the form of intact PTH.See Habener et al., Nature—New Biol., 238:152-154 (1972); Flueck et al., Journal of Clinical Research. Clin. Invest., 60:1367-1375 (1977); Mayer et al., Endocrinology, 104:1778-1784 (1979); Chu et al., Endocrinology, 93:915-924 (1973); Habener et al., Endocrinology, 97:431-441 (1975); Russell et al., Journal of Clinical Research, 72:1851-1855 (1983); Heinrich et al., Endocrinology, 112:449-458 (1983); Brookman et al., Journal of Bone and Mineral Research Bone & Min. Res., 1:529-537 (1986); Sherwood et al., Proceedings of the National Academy of Sciences of the United States of America, 67:1631-1638 (1970); Arnaud et al., American Journal of Medicine, 50:630-638 (1971); Hanley et al., Journal of Clinical Research, 62:1247-1254 (1978); Di Bella et al., Journal of Clinical Endocrinology and Metabolism (Journ. of Clin. Endocrin. & Metab., 46:604-612 (1978); Roos et al., Journal of Clinical Endocrinology and Metabolism, 53:709-721 (1981); MacGregor et al., Endocrinology, 112:1019-1025 (1983); Hanley et al., Journal of Clinical Endocrinology and Metabolism, 63:1075-1079 (1986); Morrissey et al., Endocrinology, 107:164-171 (1980); MacGregor et al., Biochemical J. Annals, 261:1929-1934 (1986); McGregor et al., Journal of Biochemistry, 254:4428-4433 (1979); Kubler et al., Experim. & Clin. Endocrin., 88:101-108 (1986); Schachter et al., Surgery, 110:1048-1052 (1991); Tanguay et al., Endocrinology, 128:1863-1868 (1991).

[0007] Furthermore, it is now apparent that the C-terminal region of PTH has novel receptors that are specific for this region of the hormone. See, e.g., Hodsman et al., Journal of Clinical Endocrinology and Metabolism, 88, pp. 5212-5220 (2003). Thus, full-length PTH has biological properties that are different from those of the N-terminal PTH analogs.

[0008] However, the importance and effects of full-length parathyroid hormone on bone growth, calcium physiology and replenishment remain poorly understood as, for example, the effects of calcium. The normal daily rise and fall in PTH levels in the blood have profound effects on bone, and in cases where bone is lost due to osteoporosis, injections of PTH can stimulate the growth of new bone.

[0009] Hypoparathyroidism is a lifelong disease characterized by insufficient production of parathyroid hormone (PTH) by the parathyroid glands. Because PTH is essential for the regulation of calcium and phosphate levels, loss of PTH decreases calcium levels in the blood and bones and increases phosphate levels (hypocalcemia and hyperphosphatemia). Hypocalcemia causes symptoms such as neuromuscular hypersensitivity, including paresthesias, muscle twitches, laryngeal spasms (which may result in the inability to speak and alert healthcare providers to an underlying medical condition, which leads to delayed or incorrect treatment), and may cause tetany and seizures.

[0010] Unlike other proteins that have been successfully formulated, PTH is particularly susceptible to various forms of degradation. For example, oxidation can occur at the methionine residues at positions 8 and 18, producing the oxidized PTH species ox-M(8)-PTH and ox-M(18)-PTH, while deamidation can occur at the asparagine at position 16, producing d16-PTH. The polypeptide chain is truncated due to the cleavage of the peptide bonds at the N-terminus and C-terminus. In addition, PTH may also adsorb to surfaces, forming nonspecific aggregates and / or precipitates, thereby reducing the available concentration of the drug. All of these degradation reactions and their combinations result in partial or complete loss of PTH biological activity. Therefore, formulations of PTH must protect against these degradation reactions.

[0011] Teriparatide (PTH(1-34), marketed by Eli Lilly and Co under the brand name calcitriol therapy for hypoparathyroidism and is able to maintain normal serum calcium levels without producing hypercalciuria.

[0012] Full-length PTH (1-84) has recently been approved as a safe and effective treatment for hypoparathyroidism (marketed by Shire-NPS Pharmaceuticals under the brand name It is the first specific hormone replacement for hypoparathyroidism and is a once-daily subcutaneous injection that can be used as an adjunct to calcium and vitamin D. However, due to its short in vivo half-life, the human pharmacokinetic profile of NATPARA exhibits rapid clearance and a high-peak-to-trough ratio, which results in significant diurnal serum calcium fluctuations and poor control of calciuria, a condition associated with elevated calcium levels in the urine.

[0013] Therefore, there is a need for improved PTH receptor agonists, particularly agonists with long-lasting activity at the PTH receptor and a more stable physiological profile. See Winer KK et al., Journal of Clinical Endocrinology and Metabolism, 2003, 88(9), 4214-20. Summary of the invention

[0014] Various non-limiting aspects and embodiments of the invention are described below.

[0015] In one aspect, parathyroid hormone (PTH) variants are provided. In some embodiments, the PTH variant according to the present invention can be a PTH-Fc fusion protein, which includes PTH or a variant thereof chemically linked to the Fc region of a human IgG antibody or a derivative thereof. In one embodiment, the PTH or a variant thereof linked to Fc can be full-length PTH (i.e., PTH (1-84)), or truncated PTH, such as C-terminal truncated PTH or a variant thereof. In one embodiment, the PTH linked to Fc can be PTH (1-74) or PTH (1-64) or PTH (1-54) or PTH (1-44). In one embodiment, the PTH linked to Fc is not PTH (1-34). In one embodiment, the PTH linked to Fc can be full-length PTH (1-84), such as a mutated PTH (1-84).

[0016] In one embodiment, the PTH linked to Fc may be any length from PTH(1-35) to PTH(1-83). In one embodiment, the PTH linked to Fc may be PTH(1-33).

[0017] In one embodiment, the PTH linked to Fc may be PTH (1-84) or PTH (1-83) or PTH (1-82) or PTH (1-81) or PTH (1-80) or PTH (1-79) or PTH (1-78) or PTH (1-77) or PTH (1-76) or PTH (1-75) or PTH (1-74) or PTH (1-73) or PTH (1-72) or PTH (1-71) or PTH (1-70) or PTH (1-69) or PTH (1-68) or PTH (1-67) or PTH (1-66) or PTH (1-65) or PTH (1-64) or PTH (1-63) or PTH (1-62) or PTH (1-61) or PTH (1 -60) or PTH (1-59) or PTH (1-58) or PTH (1-57) or PTH (1-56) or PTH (1-55) or PTH (1-54) or PTH (1-53) or PTH (1-52) or PTH (1-51) or PTH (1-50) or PTH (1-49) or PTH (1-48) or PTH (1-47) or PTH (1-46) or PTH (1-45) or PTH (1-44) or PTH (1-43) or PTH (1-42) or PTH (1-41) or PTH (1-40) or PTH (1-39) or PTH (1-38) or PTH (1-37) or PTH (1-36) or PTH (1-35) or PTH (1-33).

[0018] In one embodiment, the amino acid sequence of the PTH connected to Fc includes a further modification selected from amino acid substitution, addition or deletion. In one embodiment, the PTH connected to Fc includes F34A substitution, F34D substitution, V35S substitution or V35T substitution, or a combination thereof. In one embodiment, the PTH connected to Fc can be further modified, such as pegylation, glycosylation, etc. In one embodiment, the PTH connected to Fc is pegylated. In one embodiment, the PTH connected to Fc is glycosylated. In one embodiment, Fc is a native Fc form. In another embodiment, Fc includes a further modification selected from amino acid substitution, addition or deletion. In one embodiment, Fc is hFcLALA including L234A and L235A substitutions. In other embodiments, Fc can be modified for increasing half-life, such as described in Yang et al., mAbs, 2017, 9, 1105 and other methods known in the art.

[0019] In one embodiment, an Fc-linked PTH variant having the amino acid sequence of SEQ ID NO: 8-11 is provided.

[0020] In one embodiment, the Fc-linked PTH variant according to the present invention is provided in a bivalent structure, ie, having two copies of PTH and one copy of Fc.

[0021] In another embodiment, the Fc-linked PTH variant according to the present invention is provided in a monovalent structure, i.e., having one copy of PTH and one copy of Fc. In one embodiment, the monovalent PTH-Fc variant can be prepared using heavy chain mutations known in the art (e.g., knobs and holes, etc.). One method known in the art is described, for example, in U.S. Pat. No. 8,679,785. In another embodiment, the monovalent PTH-Fc variant can be prepared using a monomeric Fc, wherein the two Fc portions can be connected by a linker (e.g., an amino acid linker).

[0022] In one embodiment, the serum half-life of the PTH-Fc fusion protein of the present invention is longer than the serum half-life of PTH (1-84). In one embodiment, the serum half-life of the PTH-Fc fusion protein of the present invention is 2 times longer, or 3 times longer, or 5 times longer, or 10 times longer, or 20 times longer, or 30 times longer, or 40 times longer than the serum half-life of PTH (1-84). In one embodiment, the serum half-life of the PTH-Fc fusion protein of the present invention is 40 times longer than the serum half-life of PTH (1-84).

[0023] In another embodiment, the peak-to-trough ratio of the PTH-Fc fusion protein is lower than the peak-to-trough ratio of exogenous PTH (1-84). In one embodiment, the peak-to-trough ratio of the PTH-Fc fusion protein of the present invention is at least 2 times lower than the peak-to-trough ratio of PTH (1-84). In one embodiment, the peak-to-trough ratio of the PTH-Fc fusion protein of the present invention is at least 10 times lower than the peak-to-trough ratio of PTH (1-84).

[0024] In some embodiments, the PTH variant is processed from a PTH variant precursor polypeptide, which includes a signal peptide directly connected to the PTH variant. The signal peptide on the polypeptide can promote the secretion of the PTH variant from a mammalian host cell used to produce the PTH variant, wherein the signal peptide is cleaved from the PTH variant after secretion. Any number of signal peptides can be used. In one embodiment, the signal peptide can have the following sequence (IgK signal peptide): METPAQLLFLLLLWLPDTTG (SEQ ID NO: 3). In another embodiment, the signal peptide can have the following sequence (IgG heavy chain signal peptide): MEFGLSWLFLVAILKGVQC (SEQ ID NO: 4). In yet another embodiment, the signal peptide can have the following sequence (CD33 signal peptide): MPLLLLLPLLWAGALA (SEQ ID NO: 5). In a specific embodiment, a CD33 signal peptide having the sequence MPLLLLLPLLWAGALA (SEQ ID NO: 5) can provide optimal signal peptide cleavage, leaving position 1 of the PTH variant intact.

[0025] In another aspect, a pharmaceutical composition comprising at least one PTH-Fc fusion protein of the present invention and a pharmaceutically acceptable carrier is provided.

[0026] In another aspect, a pharmaceutical dosage form comprising at least one PTH-Fc fusion protein of the present invention or a pharmaceutical composition of the present invention is provided. In one embodiment, the pharmaceutical dosage form of the present invention is a liquid dosage form. In one embodiment, the pharmaceutical dosage form is suitable for administration by injection or infusion.

[0027] In another aspect, a nucleic acid encoding a PTH variant according to the present invention is provided. In one embodiment, a nucleic acid encoding a PTH-Fc fusion variant protein is provided.

[0028] In another aspect, an expression vector comprising a nucleic acid encoding a PTH variant according to the present invention is provided. In one embodiment, an expression vector comprising a nucleic acid encoding a PTH-Fc fusion variant protein is provided.

[0029] In another aspect, a host cell comprising an expression vector according to the present invention or a nucleic acid encoding a PTH variant is provided. In one embodiment, a host cell comprising an expression vector or a nucleic acid encoding a PTH-Fc fusion variant protein is provided. In one embodiment, the host cell is a prokaryotic cell, a yeast cell, an insect cell or a mammalian cell. In one embodiment, the host cell is a CHO cell.

[0030] In another aspect, a method of controlling serum calcium and phosphate levels in a subject in need thereof is provided, the method comprising administering to the subject a PTH variant of the present invention, or a pharmaceutical composition comprising a PTH variant of the present invention, or a pharmaceutical dosage form comprising a PTH variant of the present invention.

[0031] In another aspect, a method of controlling urinary calcium levels in a subject in need thereof is provided, the method comprising administering to the subject a PTH variant of the present invention, or a pharmaceutical composition comprising a PTH variant of the present invention, or a pharmaceutical dosage form comprising a PTH variant of the present invention.

[0032] In another aspect, a method of treating a subject suffering from hypoparathyroidism is provided, the method comprising administering to the subject an effective amount of a PTH variant of the present invention, or a pharmaceutical composition comprising a PTH variant of the present invention, or a pharmaceutical dosage form comprising a PTH variant of the present invention.

[0033] In another aspect, a method of treating a subject suffering from hypoparathyroidism is provided, the method comprising administering to the subject an effective amount of a PTH variant of the present invention, or a pharmaceutical composition comprising a PTH variant of the present invention, or a pharmaceutical dosage form comprising a PTH variant of the present invention.

[0034] In some embodiments, the PTH variants of the present invention are administered twice a day, or once a day, or once every two days, or once every three days, or once every 5-8 days. In one embodiment, the PTH variants of the present invention are administered once a day. In one embodiment, the PTH variants of the present invention are administered once a week. In one embodiment, the PTH variants of the present invention are administered subcutaneously.

[0035] In some embodiments, due to population differences, it may be necessary to titrate the dosage of the PTH variant of the present invention for individuals. In one embodiment, the PTH variant of the present invention is administered in an amount of about 1 μg / day to about 500 μg / day, or about 2 μg / day to about 250 μg / day, or about 5 μg / day to about 100 μg / day, or about 10 μg / day to about 80 μg / day, or about 20 μg / day to about 100 μg / day, or about 50 μg / day to about 100 μg / day, or about 50 μg / day, or about 60 μg / day, or about 70 μg / day, or about 80 μg / day, or about 90 μg / day, or about 100 μg / day.

[0036] These and other aspects of the present invention will become apparent to those skilled in the art after reading the following detailed description of the invention including the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The patent or application file contains at least one drawing executed in color. Copies of the patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0038] Figure 1 Comparison of serum concentrations over time of different C-terminally truncated PTH-Fc fusion variants following a single subcutaneous administration to rats.

[0039] Figure 2 (AB) shows serum calcium ( ) of PTH-66 (PTH(1-74)-Fc fusion) in mice after a single subcutaneous administration compared to vehicle (phosphate buffer) control. Figure 2A ) and urinary calcium ( Figure 2B ) concentration changes with time.

[0040] FIG. 3 (AB) shows the PK of different concentrations of PTH-66 (PTH(1-74)-Fc) fusion on serum calcium in TxPTx rats after a single subcutaneous injection ( Figure 3A ) and PD( Figure 3B )data.

[0041] Figure 4 (AB) shows single dose PK from TPTx rats comparing PTH-66 to PTH(1-84) ( Figure 4A ) and PD( Figure 4B ) research data.

[0042] FIG. 5 (AD) shows the multiple dose PK of PTH-66 from daily subcutaneous administration to TPTx rats ( Figure 5A ) and PD( Figure 5B -D) Research data.

[0043] Figure 6 (AB) shows the single-dose PK of PTH-66 and PTH (1-84) from wild-type cynomolgus monkeys ( Fig. 6A ) and PD( Figure 6B ) research data.

[0044] Figure 7 Shown are data from a multiple-dose PK and PD study comparing PTH-66 to vehicle following daily subcutaneous administration in wild-type cynomolgus monkeys.

[0045] Fig. 8A Depicted is the microchromatographic purification of approximately 900 mL of CM expressing PTH-66 using 2 x 5 mL Mab Select Sure columns. Figure 8B is a flow chart outlining the purification steps of PTH-66 according to an embodiment of the present invention.

[0046] Fig.9AMab select crude elution fractions of PTH-66 are depicted. Fig. 9B Depicted is Coomassie blue staining of the Mab select crude elution fraction of PTH-66.

[0047] Fig.10 Depicted is a SDS-PAGE Coomassie stain of the final PTH-66 product.

[0048] Fig.11 The PTH-66 downstream process flow is depicted.

[0049] Fig.12 is a flow chart of a process for producing PTH-66 that meets the less than 10% fragmentation target.

[0050] Fig.13A is a flow chart of the development of the polishing step for PTH-66 purification; Fig. 13B It is a flow chart of the process flow.

[0051] Fig.14A This is a high-throughput screening diagram of Capto MMC ImpRes; Fig. 14B This is a high-throughput screening diagram of CaptoAdhere ImpRes to determine the optimal binding conditions; Fig. 14C is the chemical structure of Capto MMC; Fig.14D is the chemical structure of Capto Adhere.

[0052] Fig.15 Depicted is the Capto MMC ImpRes process flow.

[0053] Fig.16 Depicted is the Capto Adhere ImpRes process flow.

[0054] Fig.17A This is the gradient elution profile of PTH-66 using Capto MMC ImpRes; Fig. 17B The figure is the overflow graph of PTH-66 using Capto AdhereImpRes.

[0055] Fig.18A Depicted is the design of downstream polishing steps for PTH-66; Fig.18B is a graph showing the robustness of a process with a stable cell based on impurity removal and process yield.

[0056] Fig.19A -F depicts the stepwise yields from the industrial pilot for purification of PTH-66 compared to the yields from process development for pilot-scale production of PTH-66. DETAILED DESCRIPTION

[0057] Detailed embodiments of the present invention are disclosed herein; however, it should be understood that the disclosed embodiments are merely examples of the present invention that can be embodied in various forms. In addition, each example given in conjunction with the various embodiments of the present invention is intended to be illustrative rather than limiting. Therefore, the specific structural and functional details disclosed herein should not be interpreted as limiting, but merely as a representative basis for teaching those skilled in the art to use the present invention in various ways.

[0058] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0059] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a method" includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those skilled in the art upon reading this disclosure.

[0060] The terms "treat" or "treatment" of a condition, disorder, or condition encompass: (1) preventing, delaying, or reducing the incidence and / or likelihood of occurrence of at least one clinical or subclinical symptom of the condition, disorder, or condition developing in a subject who may be afflicted with or susceptible to the condition, disorder, or condition but does not yet experience or display clinical or subclinical symptoms of the condition, disorder, or condition; or (2) inhibiting the condition, disorder, or condition, i.e., arresting, reducing, or delaying the development of the disease or its recurrence or at least one clinical or subclinical symptom thereof; or (3) alleviating the disease, i.e., causing regression of the condition, disorder, or condition or at least one of its clinical or subclinical symptoms. The benefit to the subject being treated is statistically significant, or at least perceptible to the patient or physician.

[0061] As used herein, "subject" or "patient" or "individual" or "animal" refers to humans, veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models of diseases (e.g., mice, rats). In preferred embodiments, the subject is a human.

[0062] As used herein, the term "effective" for dosage or amount refers to an amount of a compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject in need thereof. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include an amount of each ingredient that would be effective when administered alone. The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, the severity of the condition being treated, the specific drug or drugs employed, the mode of administration, etc.

[0063] The phrase "pharmaceutically acceptable" as used in conjunction with the compositions of the present invention refers to the molecular entities and other ingredients of such compositions that are physiologically tolerable and generally do not produce adverse reactions when administered to mammals (e.g., humans). Preferably, as used herein, the term "pharmaceutically acceptable" refers to approved by a regulatory agency of the Federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more specifically in humans.

[0064] As used herein, the terms "carrier" and "diluent" refer to pharmaceutically acceptable (e.g., safe and non-toxic for administration to humans) carriers or diluents that can be used to prepare pharmaceutical formulations. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate-buffered saline), sterile saline solutions, Ringer's solution, or dextrose solution.

[0065] As used herein, the term "fusion protein" or "chimeric protein" refers to a protein produced by the connection of two or more initially isolated proteins or portions thereof. In some embodiments, a linker or spacer will be present between each protein.

[0066] As used herein, the term "half-life" is the time required for an amount such as protein concentration or activity to decrease to half its value as measured at the beginning of a time period.

[0067] As used herein, the terms "improve," "increase," or "decrease," or grammatical equivalents, refer to values ​​relative to a baseline measurement, such as a measurement in the same individual prior to starting a treatment as described herein, or a measurement in a control subject (or multiple control subjects) in the absence of a treatment as described herein. A "control subject" is a subject having the same form of disease as the subject being treated, who is approximately the same age as the subject being treated.

[0068] As used herein, the term "in vitro" refers to events that occur in an artificial environment (eg, in a test tube or reaction vessel, in cell culture, etc.) rather than in a multicellular organism.

[0069] As used herein, the term "in vivo" refers to events that occur within a multicellular organism (e.g., humans and non-human animals). In the context of cell-based systems, the term can be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

[0070] As used herein, the term "linker" refers to an amino acid sequence in a fusion protein other than the amino acid sequence that occurs at a specific position in a natural protein or derivative, and is generally designed to be flexible or to insert a structure, such as an α-helix, between two protein parts. A linker is also referred to as a spacer. A linker or spacer generally does not have a biological function itself.

[0071] The term "polypeptide" as used herein refers to a continuous chain of amino acids linked together by peptide bonds. The term is used to refer to amino acid chains of any length, but one of ordinary skill in the art will understand that the term is not limited to long chains and may refer to a minimal chain comprising two amino acids linked together by peptide bonds. As known to those skilled in the art, polypeptides may be processed and / or modified. As used herein, the terms "polypeptide" and "peptide" are used interchangeably. The term "polypeptide" may also refer to a protein.

[0072] As used herein, the term "PTH variant" refers to any derivative of native PTH (1-84), including but not limited to PTH (1-84) derivatives comprising amino acid additions, deletions and / or substitutions, truncated (e.g., C-terminally truncated) PTH (1-84), PTH fused directly or via a linker or spacer to a peptide or protein or protein domain, and PTH post-translationally modified (e.g., glycosylated, pegylated, etc.) in any manner known in the art, or a combination thereof.

[0073] PTH

[0074] Parathyroid hormone (PTH) is a polypeptide secreted by the parathyroid glands of mammals. Native PTH is 84 amino acids long and has the following sequence (SEQ ID NO: 1):

[0075] SVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNFVALGAPLAPRDAGSQRPRKKEDNVLVESHEKSLGEADKADVNVLTKAKSQ, or

[0076]

[0077] As used herein, the term "human PTH" or "hPTH" is included in the term PTH or parathyroid hormone. Human PTH can be synthesized in vivo, recombinantly (in cells), or using standard techniques known in the art.

[0078] PTH-Fc fusion

[0079] In some embodiments, the PTH variant of the present invention is a PTH-Fc fusion protein. As used herein, "Fc" refers to the Fc region of a human IgG antibody. Fc-fusion proteins (also referred to as Fc chimeric fusion proteins, Fc-Ig, Ig-based chimeric fusion proteins, and Fc-tag proteins) are composed of the Fc domain of IgG, which is chemically linked to a target peptide or protein (e.g., PTH).

[0080] The Fc domain from any IgG can be used. As non-limiting examples, the Fc domain of IgG1 or IgG2 or IgG3 or IgG4 can be used, as well as various combinations of Fc domains derived from different IgGs, such as, for example, half IgG2 and half IgG4. In a non-limiting embodiment, the PTH-Fc fusion protein of the present invention has the Fc domain of a human IgG1 antibody.

[0081] The silent effector function can be obtained by mutations in the Fc region of the antibody, and different IgGs can have different silent subregions known to those skilled in the art. For example, for IgG1, the following silent subregions are known and have been described in the art: LALA and N297A (Strohl, W., 2009, Curr. Opin. Biotechnol. Vol. 20 (6): 685-691); and D265A (Baudino et al., 2008, J. Immunol. 181: 6664-69; Strohl, W., supra). Examples of silent Fc IgG1 antibodies include the so-called LALA mutant ("hFcLALA"), which includes L234A and L235A (EU numbering) mutations in the IgG1 Fc amino acid sequence. Another example of a silent IgG1 antibody includes the D265A mutation. Another silent IgG1 antibody includes the N297A mutation, which results in a glycosylated / aglycosylated antibody.

[0082] As used herein, IgG1 hFcLALA has the following sequence, wherein the LALA mutation is underlined (SEQ ID NO: 2):

[0083] DKTHTCPPCPAPE AAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[0084] The PTH sequence can be directly or indirectly linked to the Fc domain. In one embodiment, PTH is directly linked to the Fc domain. In other embodiments, PTH is linked to the Fc domain via an amino acid linker.

[0085] In some embodiments, the Fc region can be further modified to further extend the half-life of the fusion protein. In a non-limiting example, half-life extension technology, such as ArGenX's NHance technology, can be used in conjunction with the PTH-Fc variants of the present invention. For example, NHance technology is described in U.S. Pat. No. 8,163,881 (the contents of which are incorporated herein by reference in their entirety).

[0086] In one embodiment, the Fc-linked PTH variant according to the present invention is provided in a bivalent structure, ie, having two copies of PTH and one copy of Fc.

[0087] In another embodiment, the Fc-linked PTH variant according to the present invention is provided in a monovalent structure, i.e., having one copy of PTH and one copy of Fc. In one embodiment, the monovalent PTH-Fc variant can be prepared using heavy chain mutations known in the art (e.g., knobs and holes, etc.). One method known in the art is described, for example, in U.S. Pat. No. 8,679,785. In another embodiment, the monovalent PTH-Fc variant can be prepared using a monomeric Fc, wherein the two Fc portions can be connected by a linker (e.g., an amino acid linker).

[0088] Signal peptide and protease tags

[0089] In some embodiments, the PTH variants of the present invention are processed from a PTH variant precursor polypeptide comprising a signal peptide directly linked to the PTH variant. The signal peptide on the polypeptide can promote secretion of the PTH variant from a mammalian host cell used to produce the PTH variant, wherein the signal peptide is cleaved from the PTH variant after secretion. Any number of signal peptides can be used.

[0090] In some embodiments, the IgK leader sequence is present in the variants of the invention and has the following sequence (SEQ ID NO: 3):

[0091] METPAQLLFLLLLWLPDTTG.

[0092] In some embodiments, the IgG heavy chain signal peptide present in the variants of the invention has the following sequence (SEQ ID NO: 4):

[0093] MEFGLSWLFLVAILKGVQC.

[0094] In some embodiments, the CD33 signal peptide present in the variants of the invention has the following sequence (SEQ ID NO:5):

[0095] MPLLLLLPLLWAGALA.

[0096] In a specific example, a CD33 signal peptide having the sequence MPLLLLLPLLWAGALA (SEQ ID NO: 5) can provide optimal signal peptide cleavage, leaving position 1 of the PTH variant intact.

[0097] In some embodiments, a polyhistidine tag (HIS tag) is present in the variants of the present invention to facilitate the purification of the PTH protein using an affinity chromatography resin that recognizes and binds to the HIS tag. In one embodiment, the TEV-HIS tag is present at the C-terminus of the PTH variants of the present invention. The TEV component is a protease recognition site that allows the HIS tag to be removed after protein purification. In one embodiment, the sequence of the TEV-HIS tag is ENLYFQSHHHHHH (SEQ ID NO: 6). In one embodiment, a portion of the TEV recognition sequence ENLYFQ (SEQ ID NO: 7) is retained at the end of the protein and does not affect the activity of the protein.

[0098] Protein glycosylation

[0099] As used herein, glycosylation is a reaction in which carbohydrates (glycans) are attached to a peptide or protein. Different classes of glycans are identified, of which two are most prominent: N-linked glycans attached to nitrogen or arginine side chains and O-linked glycans attached to the hydroxyl oxygen of serine, threonine, tyrosine, hydroxylysine, or hydroxyproline side chains.

[0100] In some embodiments, amino acid substitutions that insert glycosylation sites are introduced into the PTH variants of the invention. In some embodiments, these amino acid substitutions can introduce serine moieties into the PTH variants. In some embodiments, the PTH variants of the invention can include F34A, F34D and / or V35S or V35T mutations to insert glycan sites into the PTH variants.

[0101] In some embodiments, amino acid changes can be made at F34 (e.g., F34A or F34D) and / or V35 (e.g., V35S or V35T) to minimize the proteolysis observed in this region during expression of the molecule in a specific cell type (e.g., CHO cells). F34A and F34D mutations can reduce proteolysis. The combination of F34A and V35S (or V35T) mutations reduces proteolysis to a greater extent. These mutations produce a common N-connected glycosylation site, which includes the N33 position. This results in glycosylation at the N33 position, which greatly reduces the proteolysis at this position.

[0102] In some embodiments, the insertion of a glycan site at N33 reduces protein cleavage during host cell expression. Glycans introduce additional size and volume to PTH variants. Glycan sites may also improve the solubility of PTH variants. Glycan sites may also extend the half-life (T1 / 2) of PTH variants. Glycan insertion at the site of protein mutation may also reduce the potential immunogenicity of the mutant sequence. In some embodiments, amino acid substitutions inserted into glycosylation sites can be combined with other mutations or sequence truncations or post-translational modifications of PTH variants.

[0103] In some embodiments, an amino acid change can be made at L59 (eg, L59S or L59T). Such a mutation creates a glycosylation site at position N57, which may reduce protein cleavage during host cell expression.

[0104] Other variants: In some embodiments, the introduction of amino acid substitutions into PTH variants can improve developmental characteristics. In some embodiments, these amino acid substitutions result in reduced cleavage of PTH variants during expression. In some embodiments, these amino acid substitutions result in increased solubility of PTH variants. In some embodiments, these amino acid substitutions result in glycan insertions. In some embodiments, these amino acid substitutions can introduce aspartic acid into the sequence.

[0105] In some embodiments, the PTH variants of the present invention may include a F34D mutation to reduce protein cleavage during expression. In some embodiments, a PTH variant containing F34D may be combined with other mutations or sequence truncations or post-translational modifications of the PTH variant.

[0106] Typically, suitable PTH variants have an in vivo half-life of about 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, or 48 hours. In some embodiments, the PTH variants have an in vivo half-life of 2 to 48 hours, 2 to 44 hours, 2 to 40 hours, 3 to 36 hours, 3 to 32 hours, 3 to 28 hours, 4 to 24 hours, 6 to 24 hours, and 6 to 18 hours.

[0107] List of selected parathyroid hormone variants

[0108] In the following listings and throughout this specification, the following abbreviations are used:

[0109] PTH(1-N) represents parathyroid hormone peptide residues 1 to N, where N can be 84 (for native length PTH and variants) or less than 84 (for C-terminally truncated PTH variants). For example, PTH(1-84) represents parathyroid hormone peptide residues 1 to 84 (full-length sequence); PTH(1-74) represents parathyroid hormone peptide residues 1 to 74, i.e., a C-terminally truncated variant of PTH(1-84), which is the first 74 residues of PTH, wherein the last 10 residues have been removed; PTH(1-34) represents parathyroid hormone peptide residues 1 to 34, i.e., a C-terminally truncated variant of PTH(1-84), which is the first 34 residues, wherein the last 50 residues have been removed, and so on.

[0110] Point mutations in the variant sequences are designated according to standard conventions; for example, F34A indicates that the phenylalanine amino acid at position 34 is substituted with an alanine amino acid; F34D indicates that the phenylalanine amino acid at position 34 is substituted with an aspartic acid residue, V35S indicates that the valine at position 35 is substituted with a serine, and so forth.

[0111] Δ means [deleted sequence], for example, ΔLys means the removal of a lysine residue. For the constructs included herein, the Lys removed is the C-terminal region of the Fc molecule to improve, among other things, the homogeneity of the drug product.

[0112] In one embodiment, the PTH linked to Fc can be full-length PTH(1-84), such as a mutated PTH(1-84).

[0113] In one embodiment, the PTH linked to Fc may be a truncated PTH of any length from PTH (1-35) to PTH (1-83). In one embodiment, the PTH linked to Fc may be PTH (1-33).

[0114] In some embodiments of the present invention, the PTH linked to Fc may be PTH (1-84) or PTH (1-83) or PTH (1-82) or PTH (1-81) or PTH (1-80) or PTH (1-79) or PTH (1-78) or PTH (1-77) or PTH (1-76) or PTH (1-75) or PTH (1-74) or PTH (1-73) or PTH (1-72) or PTH (1-71) or PTH (1-70) or PTH (1-69) or PTH (1-68) or PTH (1-67) or PTH (1-66) or PTH (1-65) or PTH (1-64) or PTH (1-63) or PTH (1-62) or PTH (1-61) or PTH (1-60) or PTH (1-59) or PTH (1-58) or PTH (1-57) or PTH (1-56) or PTH (1-55) or PTH (1-54) or PTH (1-53) or PTH (1-52) or PTH (1-51) or PTH (1-50) or PTH (1-49) or PTH (1-48) or PTH (1-47) or PTH (1-46) or PTH (1-45) or PTH (1-44) or PTH (1-43) or PTH (1-42) or PTH (1-41) or PTH (1-40) or PTH (1-39) or PTH (1-38) or PTH (1-37) or PTH (1-36) or PTH (1-35) or PTH (1-33).

[0115] In the following description of exemplary embodiments of the present invention, the signal peptide is marked in italics, the PTH region is marked in bold, and any mutations Underline , and any fusion sequences are marked with wavy underlines.

[0116] Molecular name 'PTH-66' (SEQ ID NO: 8)

[0117] Description: PTH(1-74)-[F34A,V35S]-hFcLALA-ΔLys

[0118] Signal peptide, PTH1-74 , mutations that insert into the glycan site ,

[0119]

[0120] Molecular name 'PTH-67' (SEQ ID NO: 9)

[0121] Description: PTH(1-64)-[F34A,V35S]-hFcLALA-ΔLys

[0122] Signal peptide, PTH1-64, Mutations that insert into glycan sites ,

[0123] Molecular name 'PTH-68' (SEQ ID NO: 10)

[0124] Description: PTH(1-54)-[F34A,V35S]-hFcLALA-ΔLys

[0125] Signal peptide, PTH1-54, Mutations that insert into glycan sites ,

[0126] Molecular name 'PTH-69' (SEQ ID NO: 11)

[0127] Description: PTH(1-44)-[F34A,V35S]-hFcLALA-ΔLys

[0128] Signal peptide, PTH1-44, Mutations that insert into glycan sites ,

[0129] Molecular name 'PTH-11' (SEQ ID NO: 12)

[0130] Description: PTH(1-84)-hFcLALA

[0131]

[0132] Other PTH variants

[0133] In one embodiment, the PTH variant according to the present invention can be conjugated to albumin or a domain of albumin directly or through a linker. In one embodiment, the PTH-albumin fusion according to the present invention can be prepared by the method described in U.S. Patent No. 7,592,010. The PTH-albumin variant can be a monovalent structure, i.e., having one copy of PTH and one copy of albumin or a domain of albumin. In one embodiment, the serum half-life of the PTH-albumin fusion protein is longer than the serum half-life of PTH (1-84). In one embodiment, the serum half-life of the PTH-albumin fusion protein of the present invention is 2 times longer, or 3 times longer, or 5 times longer, or 10 times longer, or 20 times longer, or 30 times longer, or 40 times longer than the serum half-life of PTH (1-84). In one embodiment, the serum half-life of the PTH-Fc fusion protein of the present invention is 40 times longer than the serum half-life of PTH (1-84).

[0134] In one embodiment, the PTH linked to albumin may be a mutant PTH (1-84) or PTH (1-83) or PTH (1-82) or PTH (1-81) or PTH (1-80) or PTH (1-79) or PTH (1-78) or PTH (1-77) or PTH (1-76) or PTH (1-75) or PTH (1-74) or PTH (1-73) or PTH (1-72) or PTH (1-71) or PTH (1-70) or PTH (1-69) or PTH (1-68) or PTH (1-67) or PTH (1-66) or PTH (1-65) or PTH (1-64) or PTH (1-63) or PTH (1-62) or PTH (1-61) or PTH (1-60) or PTH (1-59) or PTH (1-58) or PTH (1-57) or PTH (1-56) or PTH (1-55) or PTH (1-54) or PTH (1-53) or PTH (1-52) or PTH (1-51) or PTH (1-50) or PTH (1-49) or PTH (1-48) or PTH (1-47) or PTH (1-46) or PTH (1-45) or PTH (1-44) or PTH (1-43) or PTH (1-42) or PTH (1-41) or PTH (1-40) or PTH (1-39) or PTH (1-38) or PTH (1-37) or PTH (1-36) or PTH (1-35) or PTH (1-33).

[0135] Nucleic Acids

[0136] In another aspect, a nucleic acid comprising a sequence encoding a PTH variant described herein is provided. The sequence may be 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOS: 8-12. In some embodiments, the nucleic acid may include additional non-coding sequences. The nucleic acid may further include a specific fragment, variant or consensus sequence thereof, or a deposition vector comprising at least one of these sequences. The nucleic acid molecule may be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including but not limited to cDNA and genomic DNA obtained by cloning, produced by gene therapy (e.g., using AAV), or any combination thereof. The DNA may be triple-stranded, double-stranded or single-stranded, or any combination thereof. Any portion of at least one strand of the DNA or RNA may be a coding strand (also referred to as a sense strand), or it may be a non-coding strand (also referred to as an antisense strand).

[0137] In certain embodiments, the nucleic acid encoding the transgenic can be modified to provide an increased expression of the PTH variant encoded, which is also referred to as codon optimization. For example, the nucleic acid encoding the transgenic can be modified by changing the open reading frame of the coding sequence. As used herein, the term "open reading frame" is synonymous with "ORF", and means any nucleotide sequence that may be able to encode a protein or a part of a protein. The open reading frame usually starts with a start codon (e.g., in a standard code, it is represented as AUG for RNA molecules, and is represented as ATG for DNA molecules), and is read in a codon triplet form until the frame ends with a stop codon (e.g., in a standard code, it is represented as UAA, UGA or UAG for RNA molecules, and is represented as TAA, TGA or TAG for DNA molecules). As used herein, the term "codon" refers to a sequence of three nucleotides in a nucleic acid molecule that specifies a specific amino acid during protein synthesis; It is also referred to as a triplet or a codon triplet. For example, in the 64 possible codons in the standard genetic code, two codons GAA and GAG encode the amino acid glutamine, while codons AAA and AAG specify the amino acid lysine. In the standard genetic code, three codons are stop codons, which do not specify amino acids. As used herein, the term "synonymous codon" refers to any and all codons encoding a single amino acid. Except for methionine and tryptophan, amino acids are encoded by two to six synonymous codons. For example, in the standard genetic code, the four synonymous codons encoding the amino acid alanine are GCA, GCC, GCG and GCU, the two synonymous codons specifying glutamine are GAA and GAG, and the two synonymous codons encoding lysine are AAA and AAG.

[0138] Standard codon optimization methods can be used to modify the nucleic acid encoding the open reading frame of the PTH variant. Various commercial algorithms for codon optimization are available and can be used to implement the present invention. Generally, codon optimization does not change the encoded amino acid sequence.

[0139] Nucleotide changes can change synonymous codons within the open reading frame to be consistent with endogenous codon usage found in a particular heterologous cell selected to express the PTH variant. Alternatively or additionally, nucleotide changes can change the G+C content within the open reading frame to better match the average G+C content of the open reading frame found in the endogenous nucleic acid sequence present in the heterologous host cell. Nucleotide changes can also change multiple single nucleotide regions or internal regulatory or structural sites found in the PTH variant sequence. Thus, a variety of modified or optimized nucleotide sequences are contemplated, including but not limited to nucleic acid sequences that provide increased expression of PTH variants in prokaryotes, yeast cells, insect cells, and mammalian cells.

[0140] As described herein, the polynucleotide may further comprise additional sequences, such as coding sequences for at least one signal leader peptide or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one intron, and additional non-coding sequences, including but not limited to non-coding 5' and 3' sequences, such as transcribed non-translated sequences that play a role in transcription, mRNA processing (including splicing and polyadenylation signals) (e.g., ribosome binding and stability of mRNA); additional coding sequences encoding additional amino acids, such as amino acids that provide additional functions. Thus, the sequence encoding the PTH variant or specific portion can be fused to a marker sequence (such as a sequence encoding a peptide that facilitates purification of the fused PTH variant or a specific portion of the PTH variant fragment or portion).

[0141] In addition to the polynucleotide of the present invention, the nucleic acid may further include sequences. For example, a multiple cloning site including one or more endonuclease restriction sites may be inserted into the nucleic acid to help separate the polynucleotide. In addition, a translatable sequence may be inserted to help separate the polynucleotide of the translation of the present invention. For example, a six-histidine tag sequence provides a convenient means for purifying the protein of the present invention. Nucleic acid of the present invention (not including coding sequence) is optionally a vector, adapter or joint for cloning and / or expressing the polynucleotide of the present invention.

[0142] The coding region of the transgene may contain one or more silent mutations to optimize the codon usage of a particular cell type. For example, the codons of the PTH variant may be optimized for expression in vertebrate cells. In some embodiments, the codons of the PTH variant may be optimized for expression in mammalian cells. In some embodiments, the codons of the PTH variant may be optimized for expression in human cells. In some embodiments, the codons of the PTH variant may be optimized for expression in CHO cells.

[0143] As described in the present application, nucleic acid sequences encoding PTH variants can be molecularly cloned (inserted) into suitable vectors for propagation or expression in host cells, or in some embodiments, the entire plasmid can also be synthesized. For example, a PTH variant sequence including a signal peptide effective for secreting the PTH variant from a host cell is inserted into a suitable vector, such as a sequence selected from SEQ ID NOS: 8-12. A variety of expression vectors can be used to implement the present invention, including but not limited to prokaryotic expression vectors; yeast expression vectors; insect expression vectors and mammalian expression vectors. Exemplary vectors suitable for the present invention include but are not limited to virus-based vectors (e.g., AAV-based vectors, retrovirus-based vectors, plasmid-based vectors). In some embodiments, the nucleic acid sequence encoding the PTH variant can be inserted into a suitable vector. Generally, the nucleic acid encoding the PTH variant is operably linked to various regulatory sequences or elements.

[0144] Various regulatory sequences or elements can be incorporated into expression vectors suitable for the present invention. Exemplary regulatory sequences or elements include, but are not limited to, promoters, enhancers, repressors or inhibitors, 5' untranslated (or non-coding) sequences, introns, 3' untranslated (or non-coding) sequences.

[0145] As used herein, a "promoter" or "promoter sequence" is a DNA regulatory region that is capable of binding to an RNA polymerase in a cell (e.g., directly or through a protein or substance bound by other promoters) and initiating transcription of a coding sequence. The promoter sequence is typically bound at its 3' end through a transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements required to initiate transcription at any level. The promoter may be operably associated with or operably connected to an expression control sequence (comprising enhancer and repressor sequences) or a nucleic acid to be expressed. In some embodiments, the promoter may be inducible. In some embodiments, the inducible promoter may be unidirectional or bidirectional. In some embodiments, the promoter may be a constitutive promoter. In some embodiments, the promoter may be a hybrid promoter in which the sequence containing the transcriptional regulatory region is obtained from one source, and the sequence containing the transcriptional initiation region is obtained from a second source. Systems for linking control elements to coding sequences within a transgene are well known in the art (general molecular biology and recombinant DNA techniques are described in Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, which is incorporated herein by reference). Commercial vectors suitable for inserting transgenes for expression in a variety of host cells under a variety of growth and induction conditions are also well known in the art.

[0146] In some embodiments, specific promoters can be used to control the expression of transgenes in mammalian host cells, such as, but not limited to, the SRa-promoter (Takebe et al., Molec. and Cell. Bio. 8:466-472 (1988)), the human CMV immediate early promoter (Boshart et al., Cell 41:521-530 (1985); Foecking et al., Gene 45:101-105 (1986)), the human CMV promoter, the human CMV5 promoter, the mouse CMV immediate early promoter, the EF1-α-promoter, for liver-specific expression Hybrid CMV promoter (e.g., prepared by conjugating the CMV immediate early promoter to the transcriptional promoter element of the human alpha-1-antitrypsin (HAT) or albumin (HAL) promoter), or a promoter for liver cancer-specific expression (e.g., in which human albumin (HAL; about 1000 bp) or human alpha-1-antitrypsin (HAT, about 2000 bp) is combined with the 145-long enhancer element of the human alpha-1-microglobulin and bikaryon precursor gene (AMBP); HAL-AMBP and HAT-AMBP); SV40 early promoter region (Benoist et al., Nature 290:304-310 (1981)), Douglas fir moth (Orgyia pseudotsugata) immediate early promoter, herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA, 78:1441-1445 (1981)); or the regulatory sequence of the metallothionein gene (Brinster et al., Nature, 296:39-42 (1982)). In some embodiments, the mammalian promoter is a constitutive promoter, such as, but not limited to, the hypoxanthine phosphoribosyltransferase (HPTR) promoter, adenosine deaminase promoter, pyruvate kinase promoter, β-actin promoter, and other constitutive promoters known to those of ordinary skill in the art.

[0147] In some embodiments, specific promoters can be used to control expression of transgenes in prokaryotic host cells, such as, but not limited to, the β-lactamase promoter (Villa-Komaroff et al., Proc. Natl. Acad. Sci. USA, 75:3727-3731 (1978)); the tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)); the T7 promoter, the T3 promoter, the M13 promoter, or the M16 promoter. promoter; expression in yeast host cells, such as, but not limited to, GAL1, GAL4 or GAL10 promoter, ADH (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, glyceraldehyde-3-phosphate dehydrogenase III (TDH3) promoter, glyceraldehyde-3-phosphate dehydrogenase II (TDH2) promoter, glyceraldehyde-3-phosphate dehydrogenase I (TDH1) promoter, pyruvate kinase (PYK), enolase (ENO) or triosephosphate isomerase (TPI).

[0148] In some embodiments, the promoter may be a viral promoter, many of which are capable of regulating expression of transgenes in several host cell types, including mammalian cells. Viral promoters that have been shown to drive constitutive expression of coding sequences in eukaryotic cells include, for example, simian virus promoters, herpes simplex virus promoters, papilloma virus promoters, adenovirus promoters, human immunodeficiency virus (HIV) promoters, Rous sarcoma virus promoters, cytomegalovirus (CMV) promoters, the long terminal repeats (LTRs) of Moloney murine leukemia virus and other retroviruses, the thymidine kinase promoter of herpes simplex virus, and other viral promoters known to those of ordinary skill in the art.

[0149] In some embodiments, the gene control element of the expression vector may also include 5' non-transcribed sequences and 5' non-translated sequences related to transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, Kozak sequences, etc. Enhancer elements may be optionally used to increase the expression level of a polypeptide or protein to be expressed. Examples of enhancer elements that have been shown to work in mammalian cells include SV40 early gene enhancers (such as Dijkema et al., EMBO J. (1985) 4: 761) and enhancers / promoters derived from the long terminal repeats (LTRs) of Rous sarcoma virus (RSV) (such as Gorman et al., Proceedings of the National Academy of Sciences of the United States of America, (1982b) 79: 6777) and human cytomegalovirus (such as Bossart et al., Cell, (1985) 41: 521). The genetic control elements of the expression vector will also include 3' non-transcribed sequences and 3' non-translated sequences associated with transcription and translation termination, respectively, such as a polyadenylation (polyA) signal for stabilizing and processing the 3' end of the mRNA transcribed by the promoter. Exemplary polyA signals include, for example, rabbit β-globin polyA signal, bovine growth hormone polyA signal, chicken β-globin terminator / polyA signal, and SV40 late polyA region.

[0150] The expression vector will preferably but optionally include at least one selectable marker. In certain embodiments, the selectable marker is a nucleic acid sequence encoding a resistance gene operably connected to one or more genetic control elements to give the host cell the ability to maintain viability when growing in the presence of cytotoxic chemicals and / or drugs. In certain embodiments, a selective agent can be used to maintain the retention of the expression vector in the host cell. In certain embodiments, a selective agent can be used to prevent modification (i.e., methylation) and / or silencing of the transgenic sequence in the expression vector. In certain embodiments, a selective agent is used to maintain the episomal expression of the vector in the host cell. In certain embodiments, a selective agent can be used to promote the stable integration of the transgenic sequence into the host cell genome. In some embodiments, the agent and / or resistance gene may include, but is not limited to, methotrexate (MTX), dihydrofolate reductase (DHFR, U.S. Pat. Nos. 4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017, ampicillin, neomycin (G418), zeomycin, mycophenolic acid, or glutamine synthetase (GS, U.S. Pat. Nos. 5,122,464; 5,770,359; 5,827,739) for eukaryotic host cells; tetracycline, ampicillin, kanamycin, or chloramphenicol for prokaryotic host cells; and URA3, LEU2, HIS3, LYS2, HIS4, ADE8, CUP1, or TRP1 for yeast host cells.

[0151] The expression vector can be transfected, transformed or transduced into the host cell. As used herein, the terms "transfection", "transformation" and "transduction" all refer to the introduction of an exogenous nucleic acid sequence into a host cell. In some embodiments, the expression vector containing the nucleic acid sequence encoding the PTH Fc fusion is transfected, transformed or transduced into the host cell at the same time. In some embodiments, the expression vector containing the nucleic acid sequence encoding the PTH variant is transfected, transformed or transduced into the host cell sequentially.

[0152] Examples of transformation, transfection and transduction methods well known in the art include liposome delivery, i.e., Lipofectamine ® (Hawley-Nelson, Focus 15:73(1193)). TM(Gibco BRL) method, electroporation, the CaPO4 delivery method of Graham and van der Erb, Virology, 52:456-457 (1978), DEAE-dextran mediated delivery, microinjection, gene gun particle delivery, polyaromatic hydrocarbon mediated delivery, cation mediated lipid delivery, transduction and viral infection, such as, for example, retrovirus, lentivirus, adenovirus adeno-associated virus and baculovirus (insect cells).

[0153] Once introduced into the cell, the expression vector can be stably integrated into the genome or exist as an extrachromosomal construct. The vector can also be amplified, and multiple copies can be present or integrated in the genome. In some embodiments, the cells of the present invention can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more copies of a nucleic acid encoding a PTH variant.

[0154] Host cells

[0155] In another aspect, a host cell comprising a polynucleotide described herein is provided, eg, a polynucleotide that allows for expression of a PTH variant in a host cell. The host cell can be a mammalian cell, non-limiting examples of which include BALB / c mouse myeloma cell line (NSO / 1, ECACC No.: 85110503); human retinoblasts (PER.C6, CruCell, Leiden, The Netherlands); monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (subcloned HEK293 or 293 cells for growth in suspension culture, Graham et al., Journal of General Virology (J. Gen Virol.), 36:59, 1977); human fibrosarcoma cell line (e.g., HT1080); baby hamster kidney cells (BHK21, ATCC CCL10); Chinese hamster ovary cells (CHO, Urlaub and Chasin, Proceedings of the National Academy of Sciences of the United States of America, 77:4216, 1980), including CHO EBNA (Daramola O (Daramola O) et al., Biotechnol. Prog., 2014, 30(1):132-41) and CHO GS (Fan L et al., Biotechnol. Bioeng. 2012, 109(4):1007-15; mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2). In one embodiment, the host cell can be a Chinese hamster ovary cell.

[0156] The polynucleotide may be in an expression plasmid. The expression plasmid may have any number of origins of replication known to those of ordinary skill in the art. The polynucleotide or expression plasmid may be introduced into a host cell by any number of means known to those of ordinary skill in the art. For example, a flow electroporation system such as the MaxCyte MaxCyte or MaxCyte Transfection system, to introduce polynucleotides or expression plasmids into host cells.

[0157] In various embodiments, the host cell expresses the nucleic acid. The host cell can express the PTH variant at a level sufficient for fed-batch cell culture or other large-scale. Alternative methods for large-scale production of recombinant PTH variants include roller bottle culture, bioreactor batch culture, and perfusion. In some embodiments, the recombinant PTH variant protein is produced by cells cultured in suspension. In some embodiments, the recombinant PTH variant protein is produced by adherent cells.

[0158] Production

[0159] Recombinant PTH variants can be produced by any available means. For example, recombinant PTH variants can be recombinantly produced by utilizing a host cell system engineered to express a nucleic acid encoding a recombinant PTH variant. Alternatively or additionally, recombinant PTH variants can be produced by activating an endogenous gene. Alternatively or additionally, recombinant PTH variants can be prepared partially or completely by chemical synthesis. Alternatively, recombinant PTH variants can be produced in vivo by mRNA therapy or AAV / lentiviral gene therapy.

[0160] In some embodiments, the recombinant PTH variant is produced in a mammalian cell. Non-limiting examples of mammalian cells that can be used according to the present invention include BALB / c mouse myeloma cell line (NSO / 1, ECACC No.: 85110503); human retinoblasts (PER.C6, CruCell, Leiden, The Netherlands); monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (subcloned HEK293 or 293 cells for suspension culture growth, Graham et al., Journal of General Virology, 36:59, 1977); human fibrosarcoma cell line (e.g., HT1080); baby hamster kidney cells (BHK21, ATCC CCL 10); Chinese hamster ovary cells + / - DHFR (CHO, Ullraub and Chasin, Proceedings of the National Academy of Sciences of the United States of America, 77:4216, 1980), including CHO EBNA (Daramola O et al., Biotechnology Progress, 2014, 30(1):132-41) and CHO GS (Fan L et al., Biotechnology and Bioengineering, 2012, 109(4):1007-15; mouse Sertoli cells (TM4, Mather, Reproduction Biology, 23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals of the New York Academy of Sciences, 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatoma cell line (Hep G2).

[0161] In some embodiments, the recombinant PTH variant is produced by human cells. In some embodiments, the recombinant PTH variant is produced by CHO cells or HT1080 cells.

[0162] In certain embodiments, host cells are selected for producing cell lines based on certain preferred attributes or growth under specific conditions selected for culturing cells. It will be appreciated by those skilled in the art that such attributes can be determined based on known characteristics and / or established cell lines (i.e., characterized commercially available cell lines) or by empirical evaluation. In some embodiments, cell lines can be selected based on the ability of cell lines to grow on a feeder layer of cells. In some embodiments, cell lines can be selected based on the ability of cell lines to grow in suspension. In some embodiments, cell lines can be selected based on the ability of cell lines to grow as adherent monolayers of cells. In some embodiments, cell lines for preferential post-translational modifications (e.g., glycosylation) can be selected. In some embodiments, such cells can be used with any tissue culture container or any container treated with a suitable adhesion substrate. In some embodiments, suitable adhesion substrates are selected from collagen (e.g., collagen I, II, II, or IV), gelatin, fibronectin, laminin, vitronectin, fibrinogen, BD Matrigel TM , basement membrane matrix, dermatan sulfate proteoglycan, poly-D-lysine and / or its combination composition. In certain embodiments, the adherent host cells can be selected and modified to grow in suspension under specific growth conditions. This method of modifying the adherent cells to grow in suspension is known in the art. For example, the cells can be conditioned to grow in suspension culture by gradually removing animal serum from the growth medium over time.

[0163] Typically, cells engineered to express a recombinant PTH variant may include a transgene encoding a recombinant PTH variant as described herein. It should be understood that the nucleic acid encoding the recombinant PTH variant may contain regulatory sequences, gene control sequences, promoters, non-coding sequences and / or other suitable sequences for expressing the recombinant PTH variant. Typically, the coding region is operably linked to one or more of these nucleic acid components.

[0164] In some embodiments, a batch culture method is used to express PTH variants. In some embodiments, the batch culture duration can last for 7-14 days. In some embodiments, the batch culture can last for 14-21 days. In some embodiments, a perfusion culture method (collecting culture medium over time every day) is used to express PTH variants. In some embodiments, a pseudo-perfusion culture method is used to express PTH variants (collecting culture medium daily at a single time point and replacing it with fresh culture medium). In some embodiments, a specific feeding regimen / culture medium can be used to promote the production of optimal PTH variants (improved polysaccharides, reduced shear). In some embodiments, cell density can be controlled / maintained to promote the production of optimal PTH variants (improved polysaccharides / reduced shear).

[0165] In some embodiments, recombinant PTH variants are produced in vivo by mRNA therapy. An mRNA encoding a PTH variant is prepared and administered to a patient in need of a PTH variant. The mRNA may include a sequence corresponding to the DNA sequence of SEQ ID NO: 8-12. Various routes of administration may be used, such as injection, pulmonary aerosolization, and subcutaneous electroporation. The mRNA may be encapsulated in a viral vector or a non-viral vector. Exemplary non-viral vectors include liposomes, cationic polymers, and cubosomes.

[0166] Recovery and purification

[0167] Various methods for purifying PTH variants from cells can be used. Various methods can be used to purify or separate PTH variants produced according to the various methods described herein. In some embodiments, the expressed protein is secreted into the culture medium, and cells and other solids can therefore be removed, such as, for example, by centrifugation or filtration, as the first step of the purification process. Alternatively or additionally, the expressed protein is bound to the surface of the host cell. In this embodiment, the host cells expressing the polypeptide or protein are lysed for purification. The lysis of mammalian host cells can be achieved by any number of methods well known to those of ordinary skill in the art, including physical crushing by glass beads, using detergents and exposure to high pH conditions. In some embodiments, PTH variants can be expressed as insoluble fractions (inclusion bodies). In such embodiments, cells can be collected, for example, by centrifugation and lysed using denaturants known in the art.

[0168] PTH variants can be isolated and purified by standard methods including, but not limited to, chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), gel filtration, centrifugation or differential solubility, ethanol precipitation, or by any other available technique for protein purification. See, e.g., Scopes, Protein Purification Principles and Practice, 2nd ed., Springer-Verlag, New York, 1987; Higgins, SJ and Hames, BD (eds.), Protein Expression: A Practical Approach, Oxford Univ Press, 1999; and Deutscher, MP, Simon, MI, Abelson, JN (eds.), Guide to Protein Purification: Methods in Enzymology (Methods in Enzymology Series, Vol. 182), Academic Press, 1997, all of which are incorporated herein by reference. Particularly for immunoaffinity chromatography, protein can be separated by being attached to affinity column, and the affinity column comprises the antibody produced for this protein and fixed on fixed support.Alternately, affinity tag (such as influenza virus capsid sequence, polyhistidine or glutathione-S-transferase) can be connected to protein by standard recombinant technology, so that it is allowed to be purified by suitable affinity column.Protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), leupeptone, pepstatin or aprotinin can be added in any or all stages, so as to reduce or eliminate the degradation of polypeptide or protein during the purification process.When it is necessary to lyse cells so as to separate and purify the expressed polypeptide or protein, protease inhibitors are particularly desirable.

[0169] PTH variants or specified portions can be recovered and purified from recombinant cell culture by well-known methods, including but not limited to protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, mixed mode chromatography (e.g., MEP Hypercel TM), hydroxyapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, New York, NY (1997-2003).

[0170] Neutralization after protein A purification should be performed carefully to avoid aggregation / precipitation of the PTH-Fc fusion protein.

[0171] The PTH variants or specified portions of the present invention include naturally purified products, products of chemical synthesis processes, and products produced by recombinant technology from eukaryotic hosts (including, for example, yeast, higher plants, insects, and mammalian cells). Depending on the host and the specific PTH variant used in the recombinant production process, the PTH variants or specified portions of the present invention may be glycosylated or may be non-glycosylated.

[0172] preparation

[0173] In some embodiments, the pharmaceutical composition described herein further includes a carrier. Suitable acceptable carriers include but are not limited to water, saline solutions (e.g., NaCl), saline, buffered saline, alcohol, glycerol, ethanol, gum arabic, vegetable oils, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates (such as lactose, straight-chain starch or starch), sugars (such as mannitol, sucrose or other), glucose, magnesium stearate, talc, silicic acid, viscous paraffin, spice oils, fatty acid esters, hydroxymethyl cellulose, polyvinyl pyrrolidone, etc., and combinations thereof. If necessary, the pharmaceutical preparation can be mixed with adjuvants (e.g., diluents, buffers, lipophilic solvents, preservatives, adjuvants, lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for affecting osmotic pressure, buffers, colorants, flavoring agents and / or aromatic substances, etc.), which do not react harmfully with the active compounds or interfere with their activity. In some embodiments, a water-soluble carrier suitable for intravenous administration is used.

[0174] Pharmaceutically acceptable adjuvants are preferred. Non-limiting examples and methods for preparing such sterile solutions are well known in the art, such as, but not limited to, Gennaro, ed., Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Co. (Easton, Pennsylvania) 1990. Pharmaceutically acceptable carriers can be routinely selected, which are suitable for the administration mode, solubility and / or stability of the PTH variant compositions as known in the art or as described herein. For example, sterile saline and phosphate buffered saline of slightly acidic or physiological pH can be used. The pH buffer can be phosphate, citrate, acetate, tris (hydroxymethyl) aminomethane (TRIS), N-tris (hydroxymethyl) methyl-3-aminopropanesulfonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine (which is a preferred buffer), arginine, lysine or acetate or a mixture thereof. The preferred buffer range is pH 4-8, or pH 6.5-8, or pH 7-7.5. Preservatives can be provided in the pharmaceutical composition, such as p-cresol, m-cresol and o-cresol, methyl parahydroxybenzoate and propyl parahydroxybenzoate, phenol, benzyl alcohol, sodium benzoate, benzoic acid, benzyl benzoate, sorbic acid, propionic acid, esters of p-hydroxybenzoic acid. Stabilizers for preventing oxidation, deamidation, isomerization, racemization, cyclization, peptide hydrolysis can be provided in the pharmaceutical composition, such as, for example, ascorbic acid, methionine, tryptophan, EDTA, asparagine, lysine, arginine, glutamine and glycine. Stabilizers for preventing aggregation, fibrillation and precipitation can be provided in the pharmaceutical composition, such as sodium lauryl sulfate, polyethylene glycol, carboxymethyl cellulose, cyclodextrin. Organic modifiers for solubilizing or preventing aggregation can be provided in the pharmaceutical composition, such as ethanol, acetic acid or acetate and its salt. Isotonic agents can be provided in the pharmaceutical composition, such as salt, for example sodium chloride or carbohydrates, for example glucose, mannitol, lactose, trehalose, sucrose or its mixture.

[0175] Pharmaceutical excipients and additives that can be used in the compositions of the present invention include, but are not limited to, proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, disaccharides, trisaccharides, tetrasaccharides, and oligosaccharides; derivatized sugars, such as sugar alcohols, aldonic acids, esterified sugars, and the like; and polysaccharides or sugar polymers), which may be present alone or in combination, and may account for 1-99.99% by weight or volume alone or in combination. Exemplary protein excipients include serum albumin, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid / PTH variant components (which may also exert buffering capacity) include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like. The preferred amino acid is glycine.

[0176] Carbohydrate excipients can be used, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch, etc.; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), inositol, etc.

[0177] The PTH variant composition may also include a buffer or pH adjuster; typically, the buffer is a salt prepared from an organic acid or base. Exemplary buffers include salts of organic acids such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffer.

[0178] In addition, the PTH variant compositions of the present invention may include polymer excipients / additives such as polyvinylpyrrolidone, ficolls (a polymeric sugar), dextran (e.g., cyclodextrins such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycol, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "Tween 20" and "Tween 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

[0179] These and other known pharmaceutical excipients and / or additives suitable for the PTH variant compositions according to the present invention are known in the art, for example as listed in "Remington: The Science & Practice of Pharmacy", 21st edition, Williams & Williams, (2005) and "Physician's Desk Reference", 71st edition, Medical Economics, Montvale, New Jersey (2017) (the disclosures of which are incorporated herein by reference in their entirety). Preferred carrier or excipient materials are salts (e.g., sodium chloride), carbohydrates (e.g., mannitol), and buffers (e.g., citrate).

[0180] The pharmaceutical composition can be formulated into a liquid suitable for administration by intravenous or subcutaneous injection or infusion. The liquid may include one or more solvents. Exemplary solvents include, but are not limited to, water; alcohols, such as ethanol and isopropanol; vegetable oils; polyethylene glycol; propylene glycol; and glycerol or mixtures and combinations thereof. A water-soluble carrier suitable for intravenous administration can be used. For example, in some embodiments, the composition for intravenous administration is typically a solution in a sterile isotonic aqueous buffer. If necessary, the composition may also include a solubilizer and a local anesthetic to relieve pain at the injection site. Typically, these ingredients are provided separately or mixed together in a sealed container in a unit dose form (e.g., as a dry lyophilized powder or anhydrous concentrate) such as an ampoule or pouch indicating the amount of the active agent. In the case where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or glucose / water. In the case where the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients are mixed before administration.

[0181] As described above, the formulation may preferably comprise a suitable buffer containing saline or a selected salt, and optionally a preservation solution and a formulation containing a preservative, and a multi-purpose preservation formulation suitable for pharmaceutical or veterinary use, which includes at least one PTH variant in a pharmaceutically acceptable formulation. The preserved formulation contains at least one known preservative or optionally selected from at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrate, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkyl p-hydroxybenzoates (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof in aqueous diluents. Any suitable concentration or mixture as known in the art may be used, such as 0.001-5%, or any range or value therein, such as but not limited to 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2 , 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or value therein. Non-limiting examples include, but are not limited to, preservatives, 0.1-2% m-cresol (e.g., 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (e.g., 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% alkyl p-hydroxybenzoate (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0.75, 0.9, 1.0%), and the like.

[0182] The PTH variant can be formulated for parenteral administration and may contain sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of plant origin, hydrogenated naphthalene, etc. as common excipients. Aqueous or oily suspensions for injection can be prepared by using suitable emulsifiers or wetting agents and suspending agents according to known methods. The injection preparation can be a non-toxic, non-orally administrable diluent, such as an aqueous solution or a suspension in a sterile injectable solution or solvent. As available vehicles or solvents, water, Ringer's solution, isotonic saline, etc. are permitted; as common solvents or suspending solvents, sterile non-volatile oils can be used. For these purposes, any kind of non-volatile oils and fatty acids can be used, including natural or synthetic or semi-synthetic fatty oils or fatty acids; natural or synthetic or semi-synthetic monoglycerides or diglycerides or triglycerides. Parenteral administration is known in the art and includes, but is not limited to, conventional injection means, pneumatic needle-free injection devices as described in US Pat. No. 5,851,198, and laser perforator devices as described in US Pat. No. 5,839,446.

[0183] The pharmaceutical composition can be an extended release formulation. The pharmaceutical composition can also be formulated to be used for sustained release, extended release, delayed release or slow release PTH variants, for example, including the amino acid sequence of SEQ ID NO:8. Extended release (also referred to as controlled release and sustained release) can be provided to injectable formulations. Microspheres, nanospheres, implants, depots and polymers can be used in combination with any compound, method and formulation as described herein to provide a prolonged release profile.

[0184] The PTH variants of the present invention, for example, including the amino acid sequence of SEQ ID NO: 8, can be formulated at a concentration of about 0.001 to about 100 mg / mL, or about 0.005 to about 50 mg / mL, or about 0.007 to about 20 mg / mL, or about 0.01 to about 10 mg / mL, or about 0.05 to about 5.0 mg / mL, or about 0.07 to about 2.0 mg / mL, or about 0.1 to about 1.0 mg / mL. In one embodiment, the PTH variant can be formulated at a concentration of about 0.01 to about 10 mg / mL. In one embodiment, the PTH variant can be formulated at a concentration of about 0.1 to about 1.0 mg / mL.

[0185] The preparations and compositions comprising PTH variants may optionally further comprise an effective amount of at least one compound or protein selected from at least one of diabetes or insulin metabolism related drugs, anti-infective drugs, cardiovascular (CV) system drugs, central nervous system (CNS) drugs, autonomic nervous system (ANS) drugs, respiratory drugs, gastrointestinal (GI) tract drugs, hormone drugs, drugs for fluid or electrolyte balance, blood drugs, anti-tumor drugs, immunomodulatory drugs, eye, ear or nasal drugs, topical drugs, nutritional drugs, etc. Such drugs are well known in the art, including the formulations, indications, dosages, and administration of each described herein (see, e.g., Nursing 2001 Handbook of Drugs, 21st ed., Springhouse Corp., Springhouse, Pa., 2001; Health Professional's Drug Guide 2001, ed. Shannon, Wilson, Stang, Prentice-Hall, Inc., Upper Saddle River, NJ; Pharmacotherapy Handbook, Wells et al., eds., Appleton & Lange, Stamford, Conn., each of which is incorporated herein by reference in its entirety).

[0186] PTH variants can also be formulated into sustained-release implants for extended or sustained administration of PTH variants. Such sustained-release preparations can be in the form of patches located outside the body. Examples of sustained-release preparations include complexes of biocompatible polymers, such as poly(lactic acid), poly(lactic acid-co-glycolic acid), methylcellulose, hyaluronic acid, sialic acid, silicates, collagen, liposomes, etc. When it is necessary to provide a high local concentration of PTH variant peptide antibodies, sustained-release preparations may be of particular interest.

[0187] PTH variant compositions and formulations can be provided to patients as clear solutions or in dual vials, comprising a vial of lyophilized at least one PTH variant (e.g., comprising the amino acid sequence of SEQ ID NO: 8) or a specific portion or variant reconstituted with a second vial containing an aqueous diluent. A single solution vial or dual vial that requires reconstitution can be reused multiple times and can satisfy single or multiple cycles of patient treatment, and thus provides a more convenient treatment regimen than currently available.

[0188] PTH variant compositions and formulations can be provided to patients indirectly by providing a clear solution or a double vial to a pharmacy, clinic or other such institution and facility, the double vial including a vial of at least one PTH variant (e.g., including the amino acid sequence of SEQ ID NO: 8) lyophilized or a specific portion or variant reconstituted with a second vial containing an aqueous diluent. In this case, the volume of the clear solution can be up to 1 liter or even larger, providing a large container from which a smaller portion of the PTH variant (e.g., including the amino acid sequence of SEQ ID NO: 8) or a specific portion or variant solution can be taken out one or more times for transfer to a smaller vial and provided to their customers and / or patients by the pharmacy or clinic. Such products can include packaging materials. In addition to the information required by the regulatory agency, the packaging materials can also provide conditions under which the product can be used. The packaging materials can provide instructions for patients to reconstitute the PTH variant (e.g., including the amino acid sequence of SEQ ID NO: 8) or a specific portion or variant in an aqueous diluent to form a solution and use the solution for two vials of wet / dry products within 2-24 hours or longer.

[0189] treat

[0190] In another aspect, a method for treating a subject suffering from hypoparathyroidism and / or hypocalcemia due to hypoparathyroidism is provided, comprising treating the subject with a PTH variant of the present invention using a dosing regimen that is effective in controlling the subject's serum and urine calcium levels. PTH variants may be particularly effective in treating hypoparathyroidism and / or hypocalcemia due to hypoparathyroidism because they have a longer half-life than native PTH (1-84). A longer half-life allows daily administration of PTH variants and maintenance at more physiologically relevant levels, which allows for more sustained control of serum and urine calcium.

[0191] In some embodiments, the method is effective in controlling the serum calcium level of the subject. In one embodiment, the method is effective in increasing the serum calcium level of a subject suffering from hypoparathyroidism and / or hypocalcemia due to hypoparathyroidism compared to the serum calcium level of a subject not administered the PTH variant of the present invention. In one embodiment, the method is effective in maintaining the serum calcium level of the subject at a lower peak-to-trough ratio compared to the serum calcium level of a subject receiving exogenous native PTH (1-84). In one embodiment, the method is effective in reducing the amount of calcium supplementation for a subject suffering from hypoparathyroidism.

[0192] In some embodiments, the method is effective in controlling urinary calcium secretion in a subject. In one embodiment, the method is effective in reducing urinary calcium output to levels of healthy volunteers (reducing potential renal complications).

[0193] PTH variants, such as amino acid sequences including SEQ ID NO:8, can be administered subcutaneously or intravenously. In various embodiments, multiple administrations are performed according to the dosing regimen. As used herein, the term "QD" or "qd" refers to administration once a day, "Q2D" refers to administration once every two days, etc. "QW" refers to administration once a week. "BID" refers to administration twice a day. Administration can be, for example, BID, once a day (QD), Q2D, Q3D, Q4D, Q5D, Q6D, QW, once every 8 days, once every 9 days, once every 10 days, once every 11 days, once every 12 days, once every 13 days, once every two weeks, once every 15 days, once every 16 days, or once every 17 days, once every 3 weeks or once a month.

[0194] PTH variants (e.g., comprising the amino acid sequence of SEQ ID NO: 8) can be administered subcutaneously according to a dosage regimen between 0.001 μg and 1,000 μg, twice a day, or once a day, or once every two days, or once every 5-8 days, or once a week (QW). Alternatively, the PTH variant can be administered once every three weeks or once a month, such as for maintenance purposes.

[0195] PTH variants (e.g., comprising the amino acid sequence of SEQ ID NO: 8) can be administered subcutaneously according to a dosage regimen of about 1 μg / day to about 500 μg / day, or about 2 μg / day to about 250 μg / day, or about 5 μg / day to about 100 μg / day, or about 10 μg / day to about 80 μg / day, or about 20 μg / day to about 100 μg / day, or about 20 μg / day to about 100 μg / day, or about 50 μg / day, or about 60 μg / day, or about 70 μg / day, or about 80 μg / day, or about 90 μg / day, or about 100 μg / day once (QD). PTH variants (e.g., comprising the amino acid sequence of SEQ ID NO: 8) can be administered at a concentration of 0.001 to 1,000 μg / mL.

[0196] The above dosing regimen can be carried out for a period of one month, or two months, or six months, or one year, or two years, or five years, or longer than five years to treat hypoparathyroidism and / or hypocalcemia caused by hypoparathyroidism. The PTH variant can be administered for a duration of the subject's lifetime for maintenance.

[0197] As used herein, the term "subcutaneous tissue" is defined as a layer of loose, irregular connective tissue immediately below the skin. For example, subcutaneous administration can be performed by injecting the composition into an area including, but not limited to, the thigh area, the abdominal area, the gluteal area, or the scapular area. For such purposes, the formulation can be injected using a syringe. However, other devices for administering the formulation are available, such as an injection device (e.g., Inject-ease TM and Genject TM device); syringe pen (such as Q-Cliq TM and GenPen TM ); needle-free devices (e.g. MediJector TM and BioJector TM ); and a subcutaneous patch delivery system. In some embodiments, a PTH variant, for example, comprising the amino acid sequence of SEQ ID NO: 8, or a pharmaceutical composition containing the same, is administered intravenously.

[0198] In various embodiments, the above-described methods of treating hypoparathyroidism and / or hypocalcemia due to hypoparathyroidism are used in combination with other methods of treating hypoparathyroidism and / or hypocalcemia due to hypoparathyroidism.

[0199] Examples

[0200] The following examples illustrate certain aspects of this specification. These examples should not be interpreted as limiting, because these examples only provide specific understanding and practice of the embodiment and its various aspects.

[0201] Example 1: Production and purification of Fc-fused PTH variants (taking PTH-66 as an example)

[0202] PTH-66 was transiently expressed in CHO cells and the conditioned medium was stored at -20°C until further use. The conditioned medium was thawed in a 25°C water bath and filtered through a 0.2 μm bottle top filter unit. PTH-66 protein was purified on a protein A derived resin MabSelect SuRe according to the manufacturer's protocol (Figure 8). The MabSelect SuRe column was pre-equilibrated with PBS. The thawed conditioned medium was loaded onto the column and then washed extensively with PBS. PTH-66 protein was eluted by step elution with 100 mM sodium citrate buffer at pH 3.2. The eluted fractions were analyzed on SDS-PAGE (Figure 9). The pH of the mixed eluted fractions was raised to about pH 6 with 1 M Tris pH 10. The neutralized mixed eluted fractions were dialyzed into PTH-66 storage buffer 20 mM sodium phosphate buffer, 50 mM sodium chloride, 26 mg / ml mannitol (pH 6.0) and stored at -80°C.

[0203] Fig. 8A Depicted is the microchromatographic purification of approximately 900 mL of CM expressing PTH-66 using 2 x 5 mL Mab Select Sure columns. Figure 8B is a flow chart outlining the purification steps of PTH-66 according to an embodiment of the present invention.

[0204] Fig.9A Mab select crude elution fractions of PTH-66 are depicted. Fig. 9B Depicted is Coomassie blue staining of the Mab select crude elution fraction of PTH-66.

[0205] Fig.10 Depicted is a SDS-PAGE Coomassie stain of the final PTH-66 product. The final yield was approximately 75 mg / 900 mL cell culture medium (CM). The protein was 3 mg / mL in PTH storage buffer. The protein was aliquoted and stored at -80°C.

[0206] Example 2: Effect of C-terminal truncation of PTH-Fc fusion

[0207] PTH-Fc fusion variants including truncated PTH of varying lengths were prepared according to the methods outlined in Example 1. The following PTH-Fc fusion variants were generated: PTH-66 (PTH(1-74)-Fc), PTH-67 (PTH(1-64)-Fc), PTH-68 (PTH(1-54)-Fc), PTH-69 (PTH(1-44)-Fc), and PTH(1-34)-Fc.

[0208] Serum concentrations of PTH following a single subcutaneous treatment of Spraque-Dawley rats with truncated variants and full-length PTH(1-84)-Fc at 0.25 mg / kg were assessed using the Immunotopics Bioactivity ELISA Kit (60-3000) and the results were Figure 1 Shown in.

[0209] like Figure 1 As shown, C-terminal truncation of PTH surprisingly and unexpectedly improves the exposure and pharmacokinetics of PTH-Fc fusion.

[0210] As shown in the figure, the truncated PTH-Fc fusion variants of the present invention are superior to native length PTH(1-84)-Fc fusion and teriparatide-Fc (PTH(1-34)-Fc fusion). Even more surprisingly, PTH(1-74) has a particularly long in vivo half-life compared to native length PTH(1-84)-Fc and other analogs.

[0211] Example 3: PTH-66 in normal mice

[0212] PTH-66 was administered to normal mice at a dose of 50 nmol / kg. Samples were collected at 2, 8, 24, 48 and 72 hours after administration, and calcium concentrations were plotted and compared to phosphate buffer (vehicle) controls.

[0213] Figure 2 shows serum calcium levels ( Figure 2A ) and urine calcium levels ( Figure 2B ) as a function of hours after administration of PTH-66 (50 nmol / kg dose) in mice. Figure 2A As shown, PTH-66 elicited a prolonged increase in serum calcium compared with PTH(1-84) and vehicle controls. Figure 2B , PTH-66 showed a longer maintenance of urine calcium levels than PTH(1-84). PTH(1-84) treatment resulted in a spike in urine calcium within the first few hours, consistent with the rise in serum calcium and the transient exposure of the molecule. In contrast, despite the rise in serum calcium levels, PTH-66 showed a longer sustained control of urine calcium levels, maintaining levels close to vehicle for 12-24 hours. In summary, treatment with PTH-66 resulted in a sustained effect on calcium levels.

[0214] Example 4: PTH-66 in TxPTx rats

[0215] PTH-66 was administered to normal (intact) and thyroid-parathyroidectomized (TxPTx) rats at different doses of 5, 20 and 60 nmol / kg. Serial samples were collected at 1, 6, 12, 18, 24, 36, 48 and 72 hours after administration and compared with vehicle control.

[0216] like Figure 3A As shown in A and B, TxPTx rats treated with PTH-66 exhibited a dose-linear PK / PD response, with dose-dependent serum Ca++ levels increasing and maintained for up to 72 hours after administration. Figure 3B It was shown that TxPTx rats receiving 5 nmol / kg PTH-66 had normal serum calcium levels (which were restored to those of intact rats) 12-48 hours after administration, whereas treatment with 20 and 60 nmol / kg PTH-66 showed higher serum calcium levels compared to intact rats receiving placebo vehicle.

[0217] Figure 4 (AB) shows the single-dose PK of PTH-66 and PTH (1-84) from TPTx rats ( Figure 4A ) and PD( Figure 4B ) study data. Rat TPTx data showed a significant PK extension beyond PTH(1-84), and compared with PTH(1-84), 5nmol / kg of PTH-66 could restore serum calcium to normal levels, while PTH(1-84) showed only a very transient increase in serum calcium.

[0218] In a separate multiple-dose study, PTH-66 was administered subcutaneously daily at doses of 2 and 4 nmol / kg to normal (intact) and thyroid-parathyroidectomized (TxPTx) rats for 10 days. Samples were collected once daily prior to dosing and compared to vehicle control.

[0219] Figure 5 (AD) shows the multiple dose PK of PTH-66 from daily subcutaneous administration to TPTx rats ( Figure 5A ) and PD( Figure 5B -D) Research data. Figure 5A The PK data showed a linear PK response to dose, reaching a near steady-state level of PTH-66. Figure 5B As shown, 2 and 4 nmol / kg of PTH-66 restored and maintained serum calcium levels within the normal range (intact). Figure 5C showed that daily administration of PTH-66 also resulted in normalization of serum phosphorus levels. Figure 5D It is shown that when PTH-66 is maintained as Figure 5AWhen normal serum calcium levels were shown, this molecule also maintained normal urinary calcium levels. These data indicate that daily subcutaneous administration of PTH-66 results in complete normalization of serum calcium and phosphate, and importantly, urinary calcium.

[0220] Example 5: PTH-66 in primates

[0221] PTH-66 was administered as a single dose at a dose of 2 mmol / kg to wild-type cynomolgus monkeys, while other monkeys received a single dose of 5 mmol / kg of Natpara (PTH (1-84)). Serial samples were collected at 0, 1, 3, 6, 9, 12, 18, 24, 36, 48 and 72 hours after administration and compared with Natpara and vehicle control.

[0222] Figure 6 (AB) shows the single-dose PK of PTH-66 and PTH (1-84) from wild-type cynomolgus monkeys ( Fig. 6A ) and PD( Figure 6B ) research data.

[0223] The data in Figure 6 show that the PK extension in PTH-66 is significantly greater than that of Natpara, and that 2 nmol / kg of PTH-66 can stimulate and maintain serum calcium levels beyond those obtained with PTH (1-84). Natpara has a shorter half-life and a more transient effect on serum calcium than PTH-66.

[0224] In a separate multiple-dose study, PTH-66 was administered subcutaneously daily to wild-type cynomolgus monkeys at a dose of 1 nmol / kg for 9 days. Samples were collected daily before dosing and 24, 48, 72, and 120 hours after the last dose and compared to vehicle control.

[0225] Figure 7 Data from a multiple-dose PK and PD study of wild-type cynomolgus monkeys treated subcutaneously daily with 1 nmol / kg of PTH-66 are shown. The PK data indicate that a relatively flat PK profile of PTH-66 was obtained during the study period, resulting in increases in serum calcium and decreases in serum phosphorus compared to vehicle controls.

[0226] Example 6: PTH cAMP titer determination

[0227] Table 1 summarizes the cAMP potency (EC 50 Table 2 summarizes the relevant PK data of PTH variants.

[0228] Table 1: EC of Select PTH-Fc variants 50 data

[0229]

[0230]

[0231] Table 2: PK data of Select PTH-Fc variants

[0232]

[0233] As shown in Tables 1 and 2, all PTH-Fc samples had subnanomolar EC 50 In all cases, the compounds were active and had significantly improved PK properties relative to those of PTH1-84.

[0234] Example 6: Multimodal chromatography in the purification of PTH-66

[0235] exist Fig.11 The downstream platform shown is followed by protein A chromatography as an initial capture step. The eluate is held at a low pH of 3.5 for 1 hour for viral inactivation, then neutralized to pH 6 and frozen at -80°C. Most impurities have been substantially removed, except for sheared species and host cell proteins. The following polishing step has been developed to remove residual host cell and process impurities.

[0236] Multimodal (also known as mixed-mode) chromatography is rapidly gaining popularity in antibody purification because the media allow separation of compounds with high efficiency through a variety of interactions. Two multimodal column steps were considered in downstream polishing development: cationic Capto TM MMC ImpRes and Anion Capto TM Adhere ImpRes. However, given the complex media-specific interactions, the purification process is determined by buffer composition, ionic strength, pH, and salt type. Since it is difficult to estimate the optimal binding and elution conditions, the separation conditions for these two resins were determined by the following steps to achieve the best process performance.

[0237] Moderate fragmentation has been observed with PTH-66 (PTH(1-74)-Fc fusion) from bioreactors, despite an increased circulating half-life, which may negatively impact its potency. Addition of protease inhibitors to harvested cell cultures prior to downstream processing has been shown to prevent future product degradation. Fragmentation of recombinant proteins in cell culture may be detrimental to drug safety and potency. In downstream process development, multimodal chromatography has been used successfully to separate fragment isoforms with altered hydrophobicity or charge resulting from upstream processes from intact Fc-fusion proteins. Fig.12 A flow chart of the production process that meets the target of less than 10% fragmentation is depicted in FIG.

[0238] A comprehensive high-throughput screening (HTS) of initial operating conditions for Capto MMC and Adhere ImpRes was performed using 6 μL PreDictor 96-well plates to evaluate a wide range of parameters including salt concentration and pH (see Fig.13A and 13B ).

[0239] Separation conditions for cation exchange (cations) Capto MMC ImpRes and anion exchange (anions) Capto Adhere ImpRes were determined using a high-throughput process. Fig.14A and 14B The experimental conditions for the screening of Capto MMC ImpRes included acetate, MES and phosphate buffer systems, pH 5.0 to 7.0 and salt (NaCl) concentrations of 0 to 750 mM, a resin load of 50 g / L, and an incubation time of 1 hour. The experimental conditions for the screening of Capto Adhere ImpRes included acetate, MES, phosphate and Tris buffer systems, pH 5.0 to 8.0 and salt (NaCl) concentrations of 0 to 1500 mM, a resin load of 50 g / L, and an incubation time of 1 hour.

[0240] For Capto MMC ImpRes (a Fig. 14C The optimal binding conditions for Capto Adhere ImpRes (a cationic resin with the structure shown) have been determined to contain 50 mM MES at pH 6.0 without any salt. Fig.14D The optimal overflow conditions for the anionic resin of the structure shown) have been determined to consist of 50 mM acetate buffer at pH 5.0 without any salt.

[0241] The second step was to optimize the lab-scale process accelerated by the HTS static binding capacity results. Capto MMCImpRes was run in bind-and-elute mode to maximize impurity removal. The wash steps were investigated to reduce excessive process- and product-related impurities without unnecessary product loss ( Fig.15 The subsequent Capto Adhere ImpRes step was developed as a fast flow (FT) step, allowing the process to meet product quality standards ( Fig.16 ). MMC and Adhere benchtop confirmation runs were performed to verify process robustness with respect to impurity clearance and step yields. Quantification of PTH-Fc relied on UV280 to track the yields of MMC and Adhere.

[0242] The conditions from the high-throughput studies were successfully transferred to the bench-scale as shown in Figures 17-19. Capto MMCImpRes gradient elution trace Fig.14A Depicted in . Better resolution was achieved using a 0.66 x 10 cm (3.4 mL) column, 50 mM MES, pH 6.0, eluting with a 0 to 1 M NaCl gradient. MMC effectively provided removal of HCPs, DNA, HMW, and sheared species.

[0243] Table 3: Capto MMC ImpRes gradient elution

[0244]

[0245] The MMC wash step has been investigated to reduce other process and product related impurities. The MMC wash optimization is depicted in Table 4. The optimal wash conditions have been determined to be 6CV of 200 mM NaCl and 3CV of 250 mM NaCl.

[0246] Table 4: MMC cleaning optimization

[0247]

[0248] Down Fig. 17B The Capto Adhere ImpRes overflow demonstrated additional HCP and HMW removal as shown and in Table 5. Using a 0.66 x 10 cm (3.4 mL) column, overflowed with 50 mM sodium acetate buffer at pH 5.0, better resolution was achieved.

[0249] Table 5: Capto Adhere ImpRes overflow

[0250] Total PTH-66 Protein % Fragmentation% load 100 5.4 overflow 82 4.11

[0251] exist Fig.18A The downstream polishing step design is depicted in . The process robustness with a stable pool based on impurity removal and process yield is shown in Fig.18B and are shown in Table 6 below.

[0252] Table 6: Process robustness

[0253]

[0254] exist Fig.19A -F, the quality attributes of the pilot-scale production were characterized. As shown in these figures, the step yields of the industrial pilot (PPO), a large-scale purification, are comparable to those of the process development (PD), a small laboratory scale. By comparing these two yields, it can be concluded that the purification process has been successfully scaled up using the above parameters.

[0255] The stability of the in-process samples was determined by storing the MMC eluate at ambient temperature for 5 days and Adhere FT at 4-8°C for 4 days and then analyzing using SEC-HPLC and Intact Mass analysis.

[0256] Therefore, the Fc-fusion protein fragmentation problem has been successfully addressed using multimodal chromatography, and stepwise elution from a linear gradient has been established to achieve relatively high recoveries without compromising robust impurity clearance.

[0257] ***

[0258] Since various changes can be made to the above-mentioned subject matter without departing from the scope and spirit of the present invention, all the subject matter included in the above description or defined in the appended claims is intended to be interpreted as a description and illustration of the present invention. According to the above teachings, multiple modifications and variations of the present invention are possible. Therefore, this specification is intended to include all these replacements, modifications and variations that fall within the scope of the appended claims.

[0259] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety, as if actually set forth in this specification.

Claims

1. A parathyroid hormone (PTH)-Fc fusion protein, wherein the PTH-Fc fusion protein comprises a C-terminally mutated and truncated PTH or a variant thereof chemically linked to the Fc region of a human IgG antibody or a derivative thereof, wherein the C-terminally mutated and truncated PTH or variant thereof is selected from mutated PTH (1-74), PTH (1-64), PTH (1-54), wherein in the mutated PTH (1-74), PTH (1-64), PTH (1-54), the phenylalanine at position 34 is substituted by alanine, and the valine at position 35 is substituted by serine, and The Fc region or derivative thereof is hFcLALA, wherein in the hFcLALA, lysine at positions 234 and 235 are substituted by alanine.

2. The PTH-Fc fusion protein according to claim 1, wherein the C-terminally mutated and truncated PTH or variant thereof is PTH (1-74). The PTH-Fc fusion protein according to claim 1 , wherein the PTH or its variant is glycosylated. 4 . The PTH-Fc fusion protein according to claim 1 , wherein the PTH-Fc fusion protein is PTH-66 consisting of the amino acid sequence of SEQ ID NO:

8. 5 . The PTH-Fc fusion protein according to claim 1 , wherein the PTH-Fc fusion protein is PTH-67 consisting of the amino acid sequence of SEQ ID NO:

9. 6 . The PTH-Fc fusion protein according to claim 1 , wherein the PTH-Fc fusion protein is PTH-68 consisting of the amino acid sequence of SEQ ID NO:

10.

7. A nucleic acid encoding the PTH-Fc fusion protein according to any one of claims 1 to 6. An expression vector comprising the nucleic acid according to claim 7 .

9. A host cell comprising the nucleic acid according to claim 7 or the expression vector according to claim 8.

10. The host cell according to claim 9, wherein the host cell is a prokaryotic cell, a yeast cell, an insect cell or a mammalian cell. The host cell according to claim 9 or 10, wherein the host cell is a CHO cell. 12 . The PTH-Fc fusion protein according to claim 1 , wherein the serum half-life of the PTH-Fc fusion protein is longer than that of PTH (1-84).

13. The PTH-Fc fusion protein according to claim 1, wherein the serum half-life of the PTH-Fc fusion protein is at least 2 times longer than that of PTH (1-84).

14. The PTH-Fc fusion protein according to claim 1, wherein the serum half-life of the PTH-Fc fusion protein is at least 10 times longer than that of PTH (1-84).

15. The PTH-Fc fusion protein of claim 1, wherein the serum half-life of the PTH-Fc fusion protein is at least 20 times longer than that of PTH (1-84).

16. A pharmaceutical composition comprising the PTH-Fc fusion protein according to any one of claims 1 to 6 and 12 to 15 and a pharmaceutically acceptable carrier. 17 . A pharmaceutical dosage form comprising the PTH-Fc fusion protein according to any one of claims 1 to 6 and 12 to 15 or the pharmaceutical composition according to claim 16 .

18. The pharmaceutical dosage form according to claim 17, wherein the dosage form is a liquid dosage form suitable for administration by injection or infusion.

19. Use of the PTH-Fc fusion protein according to any one of claims 1 to 6 and 12 to 15, the pharmaceutical composition according to claim 16 or the pharmaceutical dosage form according to claim 17 or 18 for the preparation of a medicament for controlling serum calcium levels in a subject in need thereof.

20. The use according to claim 19, wherein the subject suffers from hypoparathyroidism.

21. The use according to claim 19 or 20, wherein the PTH-Fc fusion protein, the pharmaceutical composition or the pharmaceutical dosage form is administered subcutaneously.

22. The use according to claim 19 or 20, wherein the PTH-Fc fusion protein, the pharmaceutical composition or the pharmaceutical dosage form is administered once a day.

23. The use according to claim 19 or 20, wherein the PTH-Fc fusion protein, the pharmaceutical composition or the pharmaceutical dosage form is administered once a week.

24. The use according to claim 19 or 20, wherein the PTH-Fc fusion protein, the pharmaceutical composition or the pharmaceutical dosage form is provided to the subject in an amount of 20 μg to 100 μg of the PTH-Fc fusion protein per day.

25. Use of the PTH-Fc fusion protein according to any one of claims 1 to 6 and 12 to 15, the pharmaceutical composition according to claim 16 or the pharmaceutical dosage form according to claim 17 or 18 for the preparation of a preparation for treating a subject suffering from hypoparathyroidism.

26. The use according to claim 25, wherein the PTH-Fc fusion protein, the pharmaceutical composition or the pharmaceutical dosage form is administered subcutaneously.

27. The use according to claim 25 or 26, wherein the PTH-Fc fusion protein, the pharmaceutical composition or the pharmaceutical dosage form is administered once a day.

28. The use according to claim 25 or 26, wherein the PTH-Fc fusion protein, the pharmaceutical composition or the pharmaceutical dosage form is provided to the subject in an amount of 20 μg to 100 μg of the PTH-Fc fusion protein per day.

29. Use of the PTH-Fc fusion protein according to any one of claims 1 to 6 and 12 to 15, the pharmaceutical composition according to claim 16 or the pharmaceutical dosage form according to claim 17 or 18 for the preparation of a pharmaceutical composition for treating a subject suffering from hypocalcemia caused by hypoparathyroidism.

30. The use according to claim 29, wherein the PTH-Fc fusion protein, the pharmaceutical composition or the pharmaceutical dosage form is administered subcutaneously.

31. The use according to claim 29 or 30, wherein the PTH-Fc fusion protein, the pharmaceutical composition or the pharmaceutical dosage form is administered once a day.

32. The use according to claim 29 or 30, wherein the PTH-Fc fusion protein, the pharmaceutical composition or the pharmaceutical dosage form is provided to the subject in an amount of 20 μg to 100 μg of the PTH-Fc fusion protein per day.

33. The PTH-Fc fusion protein according to claim 1, the pharmaceutical composition according to claim 16, or the pharmaceutical dosage form according to claim 17 or 18, wherein the PTH-Fc fusion protein comprises one copy of PTH and one copy of Fc.

34. The PTH-Fc fusion protein according to claim 1, the pharmaceutical composition according to claim 16, or the pharmaceutical dosage form according to claim 17 or 18, wherein the PTH-Fc fusion protein comprises two copies of PTH and one copy of Fc.

35. A method for preparing a C-terminally truncated parathyroid hormone (PTH)-Fc fusion protein comprising PTH(1-74) or a variant thereof, comprising purifying the (PTH)-Fc fusion protein using a multimodal chromatography resin.

36. The method of claim 35, wherein the multimodal chromatography resin comprises a cation exchange resin and an anion exchange resin.

37. The method of claim 36, wherein the cation exchange resin is Capto MMC ImpRes.

38. The method of claim 37, wherein the (PTH)-Fc fusion protein is bound in 50 mM MES buffer at pH 6.

0.

39. The method of claim 36, wherein the anion exchange resin is Capto Adhere ImpRes.

40. The method of claim 39, wherein the (PTH)-Fc fusion protein is eluted in 50 mM acetate buffer at pH 5.

0.

41. The method of any one of claims 35 to 40, further comprising gradually eluting the (PTH)-Fc fusion protein from the multimodal chromatography resin.

42. The method of any one of claims 35 to 40, wherein the purified (PTH)-Fc fusion protein comprises less than 10% total fragmented impurities.

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