Methods for production of human recombinant arginase 1 and uses thereof

Abstract

The present invention describes methods for producing recombinant arginase enzymes, such as pegylated cobalt substituted recombinant human arginase 1. Also described are pharmaceutical compositions comprising such recombinant arginases, as well as methods of treatment and uses of such recombinant arginases.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202511445921.3 (22) Application Date 2020.08.28 (30) Priority Data 62 / 894,319 2019.08.30 US (62) Divisional Application Data 202080060999.2 2020.08.28 (71) Applicant Imedica Pharmaceuticals Company Address Stockholm, Sweden (72) Inventor Scott W. Rawlinson (74) Patent Agency Beijing Lvcheng Tongye Intellectual Property Agency Co., Ltd. 11006 Patent Attorney Xu Jinguo Wu Qichao (51) Int.Cl. C12N 9 / 78 (2006.01) C12N 9 / 96 (2006.01) C07K 19 / 00(2006.01) A61K 38 / 50(2006.01) A61K 47 / 60(2017.01) A61P 25 / 00(2006.01) A61P 35 / 00(2006.01) C12R 1 / 19(2006.01) (54) Invention Title: Method for producing recombinant human arginase 1 and its use therein (57) Abstract: This invention describes a method for producing recombinant arginases such as polyethylene glycol-modified cobalt-substituted recombinant human arginase 1. The invention also describes pharmaceutical compositions comprising such recombinant arginases, as well as treatment methods and uses of such recombinant arginases. Claims (5 pages), Description (39 pages), Sequence Listing (electronic publication), Drawings (22 pages), CN 121518448 A 2026.02.13 CN 1 21 51 84 48 A 1. A method for producing purified recombinant cobalt-substituted human arginase, wherein the recombinant human arginase (rhARG) comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1, the method comprising: a. culturing *E. coli* cells producing rhARG in a bioreactor; b. lysing the *E. coli* cells; c. removing cell debris from the lysate; d. loading the cell lysate onto a cation exchange column; e. eluting the rhARG with a high-salt solution; f. incubating the eluted rhARG with a cobalt salt to form cobalt-substituted rhARG (Co-rhARG); g. applying the Co-rhARG onto an anion exchange column and collecting the flow-through; h. Apply the flow-through to a third chromatographic column; and i. Elute the Co-rhARG from the third chromatographic column with a high-salt solution. 2. The method of claim 1, wherein up to 60 grams of rhARG per liter of cation exchange resin is added.3. The method of claim 1 or 2, wherein the rhARG is eluted from the cation exchange column using a high-salt solution with a salt concentration of up to about 0.5 M. 4. The method of claim 1 or 2, wherein the rhARG is eluted from the cation exchange column using a high-salt solution with a salt concentration of about 0.1 M. 5. The method of claim 1 or 2, wherein the rhARG is eluted from the cation exchange column using a gradient of salt concentrations from about 0.0 to about 0.5 M. 6. The method of claim 1 or 2, wherein the rhARG is eluted from the cation exchange column using a gradient of salt concentrations from about 0.0 to about 0.2 M. 7. The method of any one of claims 1 to 6, wherein the cobalt salt comprises a Co2+ salt. 8. The method of any one of claims 1 to 7, wherein the cobalt salt comprises CoCl2. 9. The method of any one of claims 1 to 8, wherein the third chromatographic column comprises a multimode chromatography (MMC) column. 10. The method according to any one of claims 1 to 9, further comprising reacting the rhARG or Co-rhARG with a polyethylene glycolation reactant to provide a polyethylene glycolated protein. 11. The method according to claim 10, wherein the polyethylene glycolated protein comprises one or more of the polyethylene glycolated amino acid residues at K16, K32, K38, K40, K47, K67, K74, K82, K87, K88, K152, K154, K171, K222, K223, K312, and K321. 12. The method of claim 11, wherein the PEGylated protein comprises one or more of the following being PEGylated: about 15% to about 60% K16, about 35% to about 80% K32, about 20% to about 85% K38, about 10% to about 60% K40, about 10% to about 60% K47, about 40% to about 90% K67, about 30% to about 95% K74, about 30% to about 98% K82, about 15% to about 65% K87, about 25% to about 70% K88, about 25% to about 85% K152, about 15% to about 65% K154, about 20% to about 75% K171, and 0% to about 30% K152. K222, 0% to approximately 35% of K223, 0% to approximately 45% of K312, and 0% to approximately 45% ofK321. 13. The method according to any one of claims 10 to 12, wherein the polyethylene glycol-modified protein comprises at least polyethylene glycol-modified amino acid residues at K16, K32, K38, K40, K47, K67, K74, K82, K87, K88, K152, K154, K171, K312, and K321. 14. The method according to any one of claims 10 to 13, wherein the polyethylene glycol-modified protein does not have polyethylene glycol-modified amino acid residues at K3, K149, K190, K195, K29, K265, and K283. 15. A method for producing purified polyethylene glycol-modified recombinant cobalt-substituted human arginase, wherein the recombinant human arginase (rhARG) comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1, the method comprising: a. culturing *E. coli* producing *rhARG* in a bioreactor; b. lysing the *E. coli* cells; c. removing cell debris from the lysate; d. loading the cell lysate onto a cation exchange column; e. eluting the *rhARG* with a high-salt solution; f. incubating the eluted *rhARG* with 10 mM CoCl2 to form cobalt-substituted *rhARG* (Co-rhARG); g. applying the Co-rhARG onto an anion exchange column and collecting the flow-through; h. applying the flow-through onto a multimode chromatography (MMC) column; i. eluting the Co-rhARG from the MMC column with a high-salt solution; j. adding a molar excess of methoxyPEG. Succinimide carboxymethyl ester; k. Removal of excess PEG. 16. A method for producing purified recombinant cobalt-substituted human arginase, wherein the recombinant human arginase (rhARG) comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1, the method comprising: a. culturing *E. coli* cells producing rhARG in a bioreactor at a pH between about 36°C and about 38°C and about 7.0 and about 7.4, with stirring and aeration; i. adjusting the temperature of the bioreactor to about 29°C; ii. inducing *E. coli* cells to produce rhARG; iii. culturing the *E. coli* cells for about 18 hours; iv. harvesting the *E. coli* cells by centrifugation; b. lysing the *E. coli* cells by high-pressure homogenization at a pH between about 7.2 and about 7.6 and at about 15°C or lower in 25 mM HEPES; c. at 15°C...Cell debris is removed from the lysate by centrifugation at a temperature lower than 10°C; the lysate is then filtered through a 0.8 μm filter followed by a 0.5 μm filter; d. The cell lysate is loaded onto a cation exchange column and washed with 25 mM HEPES at pH 7.2–7.6; e. The rhARG is eluted at room temperature with a high-salt solution containing 25 mM HEPES and 0.1 M NaCl at pH 7.2–7.6; f. The eluted rhARG is incubated with 10 mM CoCl2 at room temperature for approximately 2 to approximately 8 hours to form cobalt-substituted rhARG (Co-rhARG); i. The Co-rhARG is exchanged for 50 mM Tris at pH 8.1–8.5; g. The Co-rhARG is applied to an anion exchange column and the flow-through is collected; Claims 2 / 5 pages 3 CN 121518448 A h. Apply the flow-through to a Capto multimode chromatography (MMC) column; i. Elute the Co-rhARG from the MMC column with a high-salt solution containing 50 mM Tris and 250 mM NaCl at pH 8.1–8.5. 17. A method for producing purified recombinant polyethylene glycol-modified recombinant cobalt-substituted human arginase, wherein the recombinant human arginase (rhARG) comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1, the method comprising: a. culturing *E. coli* cells producing rhARG in a bioreactor at a pH between about 36°C and about 38°C and between about 7.0 and about 7.4, with stirring and aeration; i. adjusting the temperature of the bioreactor to about 29°C; ii. inducing *E. coli* to produce rhARG; iii. culturing the *E. coli* for about 18 hours; iv. harvesting the *E. coli* by centrifugation; b. lysing the *E. coli* cells by high-pressure homogenization at a pH between about 7.2 and about 7.6 and at a temperature of about 15°C or lower in 25 mM HEPES; c. [The text abruptly ends here, so the translation stops as well.] Cell debris was removed from the lysate by centrifugation at an even lower temperature; the lysate was then filtered through a filter of approximately 0.8 micrometers, followed by a filter of approximately 0.5 micrometers; d. The cell lysate was loaded onto a cation exchange column and washed with 25 mM HEPES at pH 7.2–7.6; e. The rhARG was eluted at room temperature with a high-salt solution containing 25 mM HEPES and 0.1 M NaCl at pH 7.2–7.6; f.Incubate the eluted rhARG with 10 mM CoCl2 at room temperature for approximately 2 to 8 hours to form cobalt-substituted rhARG (Co-rhARG); i. Exchange the Co-rhARG with 50 mM Tris, pH 8.1–8.5; g. Apply the Co-rhARG to an anion exchange column and collect the flow-through; h. Apply the flow-through to a Capto multimode chromatography (MMC) column; i. Elute the Co-rhARG from the MMC column with a high-salt solution containing 50 mM Tris and 250 mM NaCl, pH 8.1–8.5 (MMC buffer); i. Exchange the MMC buffer with 20 mM sodium phosphate, 50 mM NaCl, 1.5% glycerol, pH 7.4 (Buffer 1), and adjust the protein concentration to approximately 5.0 mg / mL; ii. Apply Buffer 1... The protein concentration is concentrated to approximately 10.0 mg / mL by exchanging buffer 2 with 0.1 M sodium phosphate, pH 8.1–8.5; j. adding an excess of methoxyPEG succinimide carboxymethyl ester with a molecular weight of approximately 5,000 Da, wherein approximately 19 moles of methoxyPEG succinimide carboxymethyl ester are added for each mole of protein, and this mixture is incubated at approximately pH 8.4 for approximately 30 minutes to approximately 4 hours; k. removing the excess PEG by exchanging buffer 2 with 20 mM sodium phosphate, 50 mM NaCl, 1.5% glycerol, pH 7.4. 18. A composition comprising Co-rhARG or Co-rhARG-PEG produced by the method according to any one of claims 1 to 17. 19. The composition of claim 18, wherein the protein is covalently linked to polyethylene glycol at one or more of K16, K32, K38, K40, K47, K67, K74, K82, K87, K88, K152, K154, K171, K222, K223, K312, and K321. 20. A composition comprising recombinant human arginase (rhARG) protein, wherein the protein comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1, wherein the protein is complexed with a non-natural metal cofactor, wherein the non-natural metal cofactor is cobalt, and wherein the protein is covalently linked to polyethylene glycol at one or more of K16, K32, K38, K40, K47, K67, K74, K82, K87, K88, K152, K154, K171, K222, K223, K312, and K321.Covalently linked to polyethylene glycol at one or more of K47, K67, K74, K82, L87, K88, K152, K154, K171, K222, K223, K312, and K321. 21. The composition according to any one of claims 18 to 20, wherein the recombinant human arginase (rhARG) comprises amino acid substitutions at positions selected from the group consisting of: H100, D123, H125, D127, D231, D233, W121, D180, S229, C302, and E255. 22. The composition of any one of claims 18 to 21, wherein the recombinant human arginase (rhARG) comprises at least one amino acid substitution selected from the group consisting of: D180S, S229C, S229G, C302F, C302I, E255Q, D180E, and S229A. 23. The composition of any one of claims 18 to 22, wherein the recombinant human arginase (rhARG) comprises at least one amino acid substitution of C302. 24. The composition of any one of claims 18 to 23, wherein the recombinant human arginase (rhARG) comprises at least two amino acid substitutions. 25. The composition of any one of claims 18 to 24, wherein the recombinant human arginase (rhARG) is a truncated arginase I protein. 26. The composition of any one of claims 18 to 25, wherein the recombinant human arginase (rhARG) further comprises an exogenous protein fragment. 27. The composition of claim 26, wherein the exogenous protein fragment comprises the Fc region of an immunoglobulin or a portion thereof. 28. The composition of any one of claims 18 to 27, wherein the specific activity of Co-rhARG-PEG is in the range of about 400 U / mg to about 700 U / mg. 29. The composition of any one of claims 18 to 28, wherein, when measured in vitro, the protein exhibits a kcat / Km value in the range of about 200 mM⁻¹ s⁻¹ to about 4,000 mM⁻¹ s⁻¹ for arginine hydrolysis at pH 7.4. 30. The composition of claim 29, wherein, when measured in vitro, the protein exhibits a kcat / Km value in the range of about 400 mM⁻¹ s⁻¹ to about 2,500 mM⁻¹ s⁻¹ for arginine hydrolysis at pH 7.4. 31. The composition according to any one of claims 18 to 30, wherein PEG:Co-rhARGThe molar ratio is in the range of about 7 mol / mol to about 15 mol / mol. 32. The composition according to any one of claims 18 to 31, wherein the free PEG concentration is less than or equal to 100 µg / mL. 33. The composition according to any one of claims 18 to 32, wherein the total cobalt content in the composition is in the range of about 9 µg / mL to about 15 µg / mL. 34. The composition according to any one of claims 18 to 33, wherein when the composition is loaded onto an imaging capillary isoelectric focusing (iCIEF), at least 9 peaks are generated, wherein peak 1 is less than 20%, peak 2 is less than 30%, peaks 3+4 are in the range of 10-30%, peak 5 is in the range of 15-30%, peak 6 is in the range of 10-25%, peak 7 is less than 25%, peak 8 is less than 15%, and peak 9 is less than 8%. 35. The composition of any one of claims 18 to 34, wherein when the composition is loaded onto icIEF, at least nine peaks are generated, wherein peak 1 is in the range of 5-7%, peak 2 is in the range of 8-11%, peaks 3+4 are in the range of 16-20%, peak 5 is in the range of 21-24%, peak 6 is in the range of 21-22%, peak 7 is in the range of 14-15%, peak 8 is in the range of 5-8%, and peak 9 is in the range of 2-3%. 36. A pharmaceutical composition comprising Co-rhARG or Co-rhARG-PEG according to any one of claims 18 to 35 and a pharmaceutical carrier. 37. The pharmaceutical composition of claim 36, wherein the composition is formulated for intravenous or subcutaneous administration. 38. The pharmaceutical composition of claim 36 or 37, wherein the composition comprises potassium phosphate, sodium chloride, and glycerol. 39. The pharmaceutical composition of any one of claims 36 to 38, wherein the composition comprises about 50 mM NaCl, about 1 mM K₂HPO₄, about 4 mM KH₂PO₄, and about 1.5% w / v glycerol. 40. A method of treating arginase 1 deficiency, the method comprising administering to a patient the pharmaceutical composition of any one of claims 36 to 39. 41. The method of claim 40, wherein the pharmaceutical composition is administered intravenously. 42. The method of claim 40, wherein the pharmaceutical composition is administered subcutaneously. 43. Claims 40 to 42The method of any one of claims 40 to 43, wherein the pharmaceutical composition is initiated at a dose of 0.1 mg / kg based on the weight of the unpolyglycolated enzyme. 44. The method of any one of claims 40 to 44, further comprising monitoring the patient's plasma arginine level. 45. The method of any one of claims 40 to 44, wherein the dose is adjusted according to the following algorithm: a. If the plasma arginine level is >150 μM, the dose is increased by two dose levels in the table below (not exceeding 0.20 mg / kg) using a single 168-hour sample, provided that the two doses prior to this sample were a) the same dose level in mg / kg and b) consecutive (without missed doses); b. If the plasma arginine levels from two consecutive 168-hour samples (regardless of whether a dose was missed) are both <50 μM, the dose is decreased by one dose level in the table below, but not to below 0.05 mg / kg. Claims 5 / 5 Page 6 CN 121518448 A Method for producing human recombinant arginase 1 and its use in the technical field

[0001] This disclosure generally relates to enzyme replacement therapy and treatment for arginase 1 deficiency or hyperarginemia. This disclosure also includes methods for producing human recombinant arginase 1. Arginase 1 can also be used to treat cancer. Background Art

[0002] Arginase 1 deficiency or hyperarginemia is a rare amino acid metabolic disorder caused by a deficiency of arginase 1. Arginase 1 is one of six enzymes essential for the normal function of the urea cycle; it catalyzes the conversion of L-arginine to urea and ornithine in the final step of the cycle. Ornithine then re-enters the mitochondria to continue the cycle.

[0003] Arginase 1 is mainly found in red blood cells (RBCs) and the liver. ARG1 is currently the only known gene whose mutations cause arginase 1 deficiency. Clinically, arginase 1 deficiency is characterized by slow degeneration of the cerebral cortex and pyramidal tract, leading to progressive dementia, psychomotor retardation, spastic paralysis, seizures, and growth retardation. If left untreated, the disease can progress to severe spasticity, inability to walk, loss of bowel and bladder control, and severe intellectual disability. Patients with arginase 1 deficiency typically have elevated blood arginine levels (3 to 4 times the upper limit of normal [ULN]), mild hyperammonemia, and a slight increase in urinary orotic acid. Most patients have no detectable arginase 1 enzyme activity in their red blood cells (<1% of normal).

[0004] Current treatment for arginase 1 deficiency focuses on reducing plasma arginine levels through lifelong dietary protein restriction.Acid concentrations are maintained at levels as close to normal as possible. Protein intake is limited to the minimum required to maintain protein biosynthesis and growth. Half or more of the dietary protein is provided in the form of a mixture of essential amino acids without arginine. This dietary adjustment can lower plasma arginine levels in most patients, but it is unpleasant, expensive, and difficult to maintain and manage, especially for growing children.

[0005] The lack of treatment options for patients with arginase 1 deficiency highlights a significant unmet need for treatments that lower arginine levels to the normal range and promote lifelong maintenance of normal arginine levels. The development of such treatments may help minimize the neurotoxic effects of patient exposure to arginine and its metabolites and offer potential for normal neurocognitive development in these patients.

[0006] In addition to treating arginase 1 deficiency or hyperarginemia, the arginase produced by these methods can be used to treat other conditions. Arginase 1 has been used in clinical trials to investigate its use in cancer treatment and in combination with immuno-oncology drugs such as pembrolizumab. Summary of the Invention

[0007] Production of Arginase

[0008] One aspect of the present invention relates to a method for producing and / or purifying recombinant human arginase protein. In one or more embodiments, the recombinant human arginase protein is recombinant human arginase 1 (rhARG1) (SEQ ID NO: 1; as shown in Figure 1(a)). In other embodiments, the recombinant human arginase protein is recombinant human arginase 2 (rhARG2) (SEQ ID NO: 3; as shown in Figure 1(c)). Although specific reference is made herein to rhARG1, the methods, formulations and uses described herein may also be applied to rhARG2. Specification 1 / 39 pages 7 CN 121518448 A

[0009] Human arginase 1 and 2 proteins subjected to the methods of the present invention have two Mn2+ sites; one or both sites may be substituted to produce arginase 1 or 2 proteins modified with non-natural metal cofactors. In some embodiments, the protein exhibits a kcat / KM greater than 200 mM⁻¹ s⁻¹ at pH 7.4. In a particular embodiment, the protein exhibits a kcat / KM in the range of about 200 mM⁻¹ s⁻¹ to about 4,000 mM⁻¹ s⁻¹ at pH 7.4. In another embodiment, the protein exhibits a kcat / KM in the range of about 400 mM⁻¹ s⁻¹ to about 2,500 mM⁻¹ s⁻¹ at pH 7.4 and 37°C. In a particular embodiment, the present invention contemplates an amino group comprising human arginase 1 or 2.Proteins with acidic sequences and non-natural metal cofactors, wherein the proteins exhibit a kcat / KM greater than 400 mM⁻¹ s⁻¹ at 37°C and pH 7.4. Exemplary kcat / KM values ​​at 37°C and pH 7.4 include about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 800, about 900, about 1,000, about 1,100, about 1,200, about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, and about 4,000 mM⁻¹ s⁻¹.

[0010] In one or more embodiments, a method for producing recombinant cobalt-substituted human arginase protein (Co-rhARG) is provided. In one or more embodiments, the method includes the following steps: fermenting *E. coli* cells expressing rhARG1; replacing manganese in rhARG1 with cobalt to provide a Co-arginase 1 intermediate (Co-rhARG1); purifying the Co-arginase 1 intermediate; and PEGylating the Co-arginase 1 intermediate to form a drug substance (Co-rhARG1-PEG). In one or more embodiments, Co-rhARG1-PEG comprises polyethylene glycol arginase.

[0011] In one or more embodiments, the method includes the following steps: culturing Escherichia coli cells in a bioreactor for producing recombinant human arginase (rhARG), lysing the E. coli cells, removing cell debris from the lysate, loading the cell lysate onto a cation exchange column, eluting the recombinant human arginase protein (rhARG) with a high-salt solution, incubating the eluted recombinant human arginase protein (rhARG) with a cobalt salt to form cobalt-substituted recombinant human arginase protein (Co-rhARG), applying the cobalt-substituted recombinant human arginase protein (Co-rhARG) to an anion exchange column and collecting the flow-through, adding the flow-through to a third chromatographic column, and eluting the cobalt-substituted recombinant human arginase protein (Co-rhARG) from the third chromatographic column with a high-salt concentration.

[0012] In one or more embodiments, a method for producing recombinant cobalt-substituted human arginase protein (Co-rhARG) includes loading up to 60 grams of recombinant human arginase protein (rhARG) per liter of cation exchange resin onto a cation exchange column.

[0013] In one or more embodiments, a method for producing recombinant cobalt-substituted human arginase protein (Co-rhARG) includes using up to about 0.5 M...A high-salt solution of high salt concentration is used to elute recombinant human arginase protein (rhARG) from a cation exchange column. In some embodiments, a 0.1 M high-salt solution is used to elute recombinant human arginase protein (rhARG) from a cation exchange column. In some embodiments, a gradient of salt concentration from about 0.0 to about 0.5 M is used to elute recombinant human arginase protein (rhARG) from a cation exchange column. In some embodiments, a gradient of salt concentration from about 0.0 to about 0.2 M is used to elute recombinant human arginase protein (rhARG).

[0014] In one or more embodiments, a method of producing recombinant cobalt-substituted human arginase protein (Co-rhARG) includes incubating the recombinant human arginase protein (rhARG) eluted from a cation exchange column with a cobalt salt containing Co2+. In some embodiments, the cobalt salt comprises CoCl2.

[0015] In one or more embodiments, a method for producing recombinant cobalt-substituted human arginase protein (Co-rhARG) includes adding a flow of recombinant cobalt-substituted human arginase protein (Co-rhARG) to a third chromatographic column comprising a multimode chromatography (MMC) column. Specification 2 / 39 pages 8 CN 121518448 A

[0016] In one or more embodiments, a method for producing recombinant cobalt-substituted human arginase protein (Co-rhARG) includes reacting recombinant cobalt-substituted human arginase protein (Co-rhARG) or recombinant cobalt-substituted human arginase protein (Co-rhARG) with a polyethylene glycolation reactant to provide a polyethylene glycolated protein. In some embodiments, the polyethylene glycol-modified protein contains one or more polyethylene glycol-modified amino acid residues at K16, K32, K38, K40, K47, K67, K74, K82, K87, K88, K152, K154, K171, K222, K223, K312, and K321. In some embodiments, the polyethylene glycol-modified protein comprises one or more of the following being polyethylene glycol-modified: about 15% to about 60% of K16, about 35% to about 80% of K32, about 20% to about 85% of K38, about 10% to about 60% of K40, about 10% to about 60% of K47, about 40% to about 90% of K67, about 30% to about 95% of K74, about 30% to about 98% of K82, about 15% to about 65% of K87, about 25% to about 70% of K88, about 25% to about 85% of K152, about 15% to about 65% of K154, about 20% to about 75% of K171, and 0% to about 30% of K38.K222, 0% to about 35% of K223, 0% to about 45% of K312, and 0% to about 45% of K321. In some embodiments, the polyethylene glycol-modified protein contains polyethylene glycol-modified amino acid residues at at least K16, K32, K38, K40, K47, K67, K74, K82, K87, K88, K152, K154, K171, K312, and K321. In some embodiments, the polyethylene glycol-modified protein does not have polyethylene glycol-modified amino acid residues at K3, K149, K190, K195, K29, K265, and K283.

[0017] One or more embodiments of Co-rhARG1-PEG involve cobalt-substituted PEGylated human recombinant arginase 1 expressed in *E. coli*, formulated for intravenous (IV) or subcutaneous (SC) administration. Replacing native manganese (Mn2+) with cobalt (Co2+) at the active site of arginase 1 enhances stability and catalytic activity at physiological pH. PEGylation also prolongs the cycling half-life (t1 / 2) of recombinant arginase 1.

[0018] In various embodiments, the method includes culturing *E. coli* cells in a bioreactor to produce recombinant human arginase 1, lysing the *E. coli* cells, and purifying the recombinant human arginase 1 (see Figures 2 and 3). Purification of the Co-arginase 1 intermediate can be performed via a purification procedure comprising one or more of the following steps: cell disruption by high-pressure homogenization, homogenization clarification, SP Sepharose FF cation exchange capture chromatography, cobalt exchange, ultrafiltration / percolation, Q Sepharose FF anion exchange flow chromatography, Capto MMC multimode chromatography, and ultrafiltration / percolation. The purified Co-arginase 1 intermediate can be processed to form a polyethylene glycol-modified drug substance or frozen and stored for later conversion into a drug substance.

[0019] In a preferred embodiment of the method, E. coli lysate containing rhARG1 is loaded onto a cation exchange (CEX) column (also referred to as “Column 1”) to capture rhARG1, and then eluted with a high-salt solution to provide a first protein product (“first protein product”).

[0020] In one or more embodiments, the method further comprises loading the first protein product onto an anion exchange (AEX) column (also referred to as “Column 2”) and collecting the flow to provide a second protein product (“second protein product”). In another aspect of the method, the method further includes loading a second protein product onto a multimode chromatography (MMC) column, which captures arginase 1 and then elutes to provide a third protein product (“third protein product”). In some embodiments, this third chromatographic column (also referred to as a “column”)3) can be a size exclusion chromatography (SEC) column.

[0021] Various embodiments include replacing the natural manganese coenzyme of arginase with a cobalt coenzyme. Cobalt substitution (also known as cobalt loading) can be performed at any step of the manufacturing process. For example, cobalt loading of arginase 1 can be performed on E. coli lysate, a first protein product, a second protein product, a third protein product, or on polyethylene glycol-modified arginase 1 at any step. In other embodiments, cobalt loading can be performed on arginase 1 eluted from column 1, or arginase 1 eluted from column 2, or arginase 1 eluted from column 3. Cobalt loading can be performed on arginase 1 eluted from a CEX column, arginase 1 eluted from an AEX column, arginase 1 eluted from an MMC column, or arginase 1 eluted from an SEC column.

[0022] Arginase 1 Cobalt loading can be performed using various cobalt-containing solutions and at various temperatures. In one or more embodiments, the cobalt salt includes Co2+ such as CoCl2. In a preferred embodiment, cobalt loading of arginase 1 is performed with CoCl2 at or around room temperature, such as at about 15 to about 25°C or about 20 to about 25°C. The cobalt loading rate can be controlled by increasing or decreasing the reaction temperature. Cobalt loading can also be performed within a certain pH range.

[0023] One aspect of this disclosure relates to changing the conditions associated with CEX chromatography (column 1). The amount of protein loaded onto column 1 can be increased or decreased to select different charge variants of arginase 1. The loading factor can be manipulated to induce a shift toward a more desirable CEX charge class distribution. Loading factors up to about 60 g / L (grams of protein / liters of CEX column resin) can produce arginase 1 with high specific activity. In various embodiments, loading factors up to about 10 g / L, about 20 g / L, about 30 g / L, about 40 g / L are used. g / L, about 50 g / L, or about 60 g / L.

[0024] In a preferred embodiment, arginase 1 is first captured on column 1, then purified sequentially on column 2 and then on column 3. In an alternative embodiment, E. coli lysate can be loaded onto an AEX column (e.g., column 2) and flow-through applied to a CEX column to capture arginase 1. In another embodiment, cobalt loading of arginase 1 can occur after polyethylene glycolation. Furthermore, other chromatographic columns can be used to replace MMC columns, such as SEC columns.

[0025] One aspect of the invention relates to a method for producing recombinant cobalt-substituted human arginase protein (Co-rhARG).Method. In one or more embodiments, the recombinant human arginase protein (rhARG) comprises an amino acid sequence that is at least 98% identical to that of SEQ ID NO:1. The method comprises several steps: culturing *E. coli* cells in a bioreactor for producing recombinant human arginase (rhARG); lysing the *E. coli* cells; removing cell debris from the lysate; loading the cell lysate onto a cation exchange column; eluting the recombinant human arginase protein (rhARG) with a high-salt solution; incubating the eluted recombinant human arginase protein (rhARG) with a cobalt salt to form cobalt-substituted recombinant human arginase protein (Co-rhARG); applying the cobalt-substituted recombinant human arginase protein (Co-rhARG) to an anion exchange column and collecting the flow-through; adding the flow-through to a third column; eluting the cobalt-substituted recombinant human arginase protein (Co-rhARG) from an MMC column with a high-salt solution; reacting with a molar excess of methoxyPEG succinimide carboxymethyl ester; and removing excess PEG.

[0026] Recombinant human arginase 1, pharmaceutical compositions and formulations

[0027] Another aspect of the invention relates to rhARG1, Co-rhARG1 and / or Co-rhARG1-PEG, or compositions comprising them, produced by the methods described herein.

[0028] In one or more embodiments, the protein is covalently linked to polyethylene glycol at one or more of K16, K32, K38, K40, K47, K67, K74, K82, L87, K88, K152, K154, K171, K222, K223, K312 and K321.

[0029] Another aspect of the invention relates to a composition comprising recombinant human arginase (rhARG) protein, wherein the protein comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1, wherein the protein is complexed with a non-natural metal cofactor, wherein the non-natural metal cofactor is cobalt, and wherein the protein is covalently linked to polyethylene glycol at one or more of K16, K32, K38, K40, K47, K67, K74, K82, K87, K88, K152, K154, K171, K222, K223, K312 and K321.

[0030] In one or more embodiments, the protein comprises amino acid substitutions at positions selected from the group consisting of: H100, D123, H125, D127, D231, D233, W121, D180, S229, C302, and E255.

[0031] In one or more embodiments, the protein comprises at least one amino acid substitution selected from the group consisting of:Substitutions: D180S, S229C, S229G, C302F, C302I, E255Q, D180E, and S229A.

[0032] In one or more embodiments, the at least one amino acid is substituted with C302. Specification 4 / 39 pages 10 CN 121518448 A

[0033] In one or more embodiments, the protein comprises at least two amino acid substitutions.

[0034] In one or more embodiments, the protein is a truncated arginase I protein.

[0035] In one or more embodiments, the protein further comprises an exogenous protein fragment.

[0036] In one or more embodiments, the exogenous protein fragment comprises the Fc region of an immunoglobulin or a portion of the Fc region of an immunoglobulin.

[0037] In one or more embodiments, the specific activity of Co-rhARG-PEG is in the range of about 400 U / mg to about 700 U / mg.

[0038] In one or more embodiments, when measured in vitro, the protein exhibits a kcat / Km value in the range of about 200 mM⁻¹ s⁻¹ to about 4,000 mM⁻¹ s⁻¹ for arginine hydrolysis at pH 7.4.

[0039] In one or more embodiments, when measured in vitro, the protein exhibits a kcat / Km value in the range of about 400 mM⁻¹ s⁻¹ to about 2,500 mM⁻¹ s⁻¹ for arginine hydrolysis at pH 7.4.

[0040] In one or more embodiments, the molar ratio of PEG:Co-rhARG is in the range of about 7 mol / mol to about 15 mol / mol.

[0041] In one or more embodiments, the free PEG concentration is less than or equal to 100 μg / mL.

[0042] In one or more embodiments, the total cobalt content of the composition is in the range of about 9 μg / mL to about 15 μg / mL.

[0043] In one or more embodiments, when the composition is loaded onto an imaging capillary isoelectric focusing (iCIEF), at least nine peaks are generated, wherein peak 1 is less than 20%, peak 2 is less than 30%, peak 3+4 is in the range of 10-30%, peak 5 is in the range of 15-30%, peak 6 is in the range of 10-25%, peak 7 is less than 25%, peak 8 is less than 15%, and peak 9 is less than 8%.

[0044] In one or more embodiments, when the composition is loaded onto an iCIEF, at least nine peaks are generated, wherein peak 1 is in the range of 5-7%, peak 2 is in the range of 8-11%, peak 3+4 is in the range of 16-20%, and peak 5 is in the range of 21-24%.Within the range, peak 6 is in the range of 21-22%, peak 7 is in the range of 14-15%, peak 8 is in the range of 5-8%, and peak 9 is in the range of 2-3%.

[0045] Another aspect of the invention relates to a pharmaceutical composition comprising rhARG1, Co-rhARG1 and / or Co-rhARG1-PEG and a pharmaceutical carrier. In one or more embodiments, the composition is formulated for intravenous or subcutaneous administration. In one or more embodiments, the composition comprises potassium phosphate, sodium chloride and glycerol. In one or more embodiments, the composition comprises about 50 mM NaCl, about 1 mM K2HPO4, about 4 mM KH2PO4 and about 1.5% w / v glycerol.

[0046] Method of treating arginase 1 deficiency

[0047] Another aspect of the invention relates to the administration of recombinant human arginase 1 such as Co-rhARG1-PEG. Such administration can be carried out by any suitable method, including IV or SC administration. In one or more embodiments of this aspect, the dose of Co-rhARG1-PEG is determined by a specific algorithm:

[0048] In one or more embodiments of this algorithm, the patient begins treatment with 0.10 mg / kg. Plasma arginine levels are monitored. If the plasma arginine level is > 150 μM, the dose is increased to 0.20 mg / kg. If the plasma arginine level is < 50 μM, the dose is reduced to 0.05 mg / kg. Otherwise, the patient maintains a dose of 0.10 mg / kg.

[0049] In one or more embodiments of the algorithm, the dose is modified as follows:

[0050] • If the plasma arginine level is >150 μM, the dose will be increased by two dose levels in the table below (not exceeding 0.20 mg / kg) using a single 168-hour sample, provided that the two doses preceding this sample are a) the same dose level calculated as mg / kg on page 5 / 39 of the specification (CN 121518448 A) and b) consecutive (without missed doses).

[0051] • If the plasma arginine levels from two consecutive 168-hour samples (regardless of whether there are missed doses) are both <50 μM, the dose will be decreased by one dose level in the table below, but not to below 0.05 mg / kg.

[0052] Brief Description of the Drawings

[0053] Other features of the invention will become apparent from the following written description and drawings, in which:

[0054] Figure 1 shows the amino acid and DNA sequence of arginase 1 and the amino acid sequence of arginase 2. Figure 1(a)Figure 1(b) shows the amino acid sequence of recombinant human arginase 1 expressed in *E. coli* (SEQ ID NO: 1); and Figure 1(c) shows the codon-optimized DNA sequence of recombinant human arginase 1 (SEQ ID NO: 2). The expressed arginase 1 monomer lacks the N-terminal methionine found in the native human arginase 1 monomer. Figure 1(c) shows the amino acid sequence of arginase 2, which lacks the N-terminal methionine found in the native human arginase 2 monomer.

[0055] Figure 2 is a schematic diagram of an exemplary process of *E. coli* fermentation and arginase 1 expression.

[0056] Figure 3 is a schematic diagram of an exemplary process for purifying recombinant human arginase 1, cobalt-substituted recombinant human arginase 1, and polyethylene glycol-modified cobalt-substituted recombinant human arginase 1. Figure 3(a) shows an exemplary process including a cation exchange column (column 1), an anion exchange column (column 2), and a Capto multimode column (column 3), as well as a cobalt loading step. Figure 3(b) shows the polyethylene glycolation of the Co-arginase 1 intermediate, followed by final filtration and formulation to provide the active pharmaceutical ingredient.

[0057] Figure 4 shows the column chromatography purification of arginase 1. Figure 4(a) shows loading E. coli cell lysate onto a cation exchange column (column 1), washing the column, and then eluting with a high-salt solution (to provide the first protein product). Protein loading and elution were evaluated by measuring UV absorbance at 280 nm. Approximately 3 liters (L) of cell lysate were applied to the column, followed by washing the column with approximately 1.5 L of buffer, and then eluting with less than approximately 1 L. Figure 4(b) shows loading arginase 1 (the first protein product) eluted from column 1 onto an anion exchange column (column 2), with protein concentration measured using absorbance at 280 nm. Arginase 1 was collected from the flow-through from column 2 to provide the second protein product. Figure 4(c) shows the capture of arginase 1 onto a Capto multimode cation exchange column (column 3) and elution with a high-salt solution to provide a third protein product.

[0058] Figure 5 shows the results of an analytical cation exchange HPLC method used to determine the charge heterogeneity distribution of the Co-arginase 1 intermediate sample (also known as the first protein product) eluted from column 1. A 1 mg / ml arginase 1 sample was loaded onto the cation exchange column with a mobile phase of 20 mM MES buffer, pH 6.0, at a flow rate of 1.0 mL / min. A 0–500 mM NaCl gradient was introduced over 40 min, and the amount of protein eluted from the column was estimated by the absorbance at 280 nm. Figure 5(a)A representative chromatogram of the charge heterogeneous substances of arginase 1 is shown. After 10–20 minutes, the charge variants of arginase 1 eluted from the analytical HPLC column. Figure 5(b) shows the same chromatogram as Figure 5(a) on page 6 / 39 of the specification, but with a greater peak magnification. Figure 5(c) shows the peak numbers assigned to the cation-exchange charge variants of arginase 1. Figure 5(d) shows the typical charge heterogeneity distribution of the active pharmaceutical ingredient as resolved by the imaging capillary isoelectric focusing (iCIEF) method.

[0059] Figure 6 shows the results of the LC / MS method used to identify the glucosylated variants of arginase 1 produced by expressing rhARG in Escherichia coli. LC / MS analysis identified unmodified arginase 1 (monomer), glucosylated arginase 1, phosphoglucosylated arginase 1, and 2-fold (2X) glucosylated arginase 1. The traces were obtained from two independent production runs of the drug intermediate. Mass spectra were superimposed on RP LCMS at 35°C for 33–35 minutes; the spectra were normalized to the peak intensity of the signal produced by the unmodified arginase 1. The peak intensity of the variant was proportional to its relative abundance.

[0060] Figure 7 shows the results of applying a 0.0–0.2 M NaCl gradient to column 1. Fractions were collected per 0.25 CV (column volume) using this gradient. The data represent two column 1 runs using two different batches of harvested cytoplasm as feed. The loading factor used for evaluation was 30 g / L. This gradient successfully separated the different glucosinated substances while maintaining product recovery.

[0061] Figure 8 shows the enzyme activity of the Co-arginase 1 intermediate and the Co-rhARG1-PEG active pharmaceutical ingredient. Figure 8(a) shows a representative enzyme kinetic analysis of the Co-arginase 1 intermediate (conversion of arginine to ornithine at substrate concentrations ranging from 0 to 2 mM at 37°C). Figure 8(b) shows a representative enzyme kinetic analysis of the Co-rhARG1-PEG active pharmaceutical ingredient.

[0062] Figure 9 shows a pharmacokinetic analysis of the Co-rhARG1-PEG active pharmaceutical ingredient. Figures 9(a) and (b) show the mean (±SD) arginase 1 concentration versus time curves in patients after a single IV dose of Co-rhARG1-PEG: Part 1. Linear plot (a) and semi-logarithmic plot (b) are shown. Note that the first mean BQL concentration is plotted at half of LLOQ (0.125 µg / mL). The mean circulating drug concentration in all patients is shown with the Co-rhARG1-PEG dose.The concentration increases with increasing dosage. Figures 9(c) through 9(f) show the mean (±SD) Co-rhARG1-PEG concentration versus time curves in patients after QW (weekly) IV dose administration of Co-rhARG1-PEG: Part 2. Linear plots for week 1 (c) and week 8 (d); semi-logarithmic plots for week 1 (e) and week 8 (f).

[0063] Figure 10 shows three representative composite plots (a, b, c) of pharmacokinetic (PK) and pharmacodynamic (PD) in a phase 1 / 2 open-label study to evaluate Co-rhARG1-PEG administration in patients with arginase 1 deficiency. Using the incremental stop criteria, the doses determined in Part 2 were: 0.09 mg / kg for patient 1, 0.12 mg / kg for patient 3, and 0.04 mg / kg (for the period shown in Part 2). By applying dose escalation cessation criteria, other patients in the trial determined the various dose levels used in Part 2. These same criteria can be used to adjust (increase or decrease) the dose of any patient already taking Co-rhARG1-PEG as a response to arginine levels outside the preferred (healthy) range.

[0064] Figure 11 shows a comparison of IV and subcutaneous administration of Co-rhARG1-PEG. The preferred plasma arginine concentration for patients is between 40 μM and 115 μM (dashed line). Subcutaneous administration of Co-rhARG1-PEG resulted in arginine concentrations remaining within this preferred range for a longer period than with IV administration. Figure 11(a) includes data from the first week after the end of Part 2, while Figure 11(b) does not include IV data from the extended period of week 1. The figures are shown as averages of patient values, and the data are from the dose determined for each patient based on the cessation criteria.

[0065] Figure 12 shows plasma arginine and guanidine compound levels after Co-rhARG1-PEG administration. Figure 12(a) shows plasma arginine levels at baseline, after dose 1, after dose 8, and during open-label extension (OLE). Figure 12(b) shows plasma levels of guanidinoacetic acid (GAA), N-α-acetyl-arginine (NAA), α-keto-δ-guanidinopentanoic acid (GVA), and arginine (ARGA) at baseline and during OLE.

[0066] Figure 13 shows baseline defects and clinical response outcomes. Figure 13(a) shows the 6-minute walk test (6MWT), gross motor function measurement (GMFM) D, and E.Figure 13(b) shows the clinical responses to 6MWT, GMFM-D, and GMFM-E.

[0067] Figure 14 shows the time-dependent improvements of 6MWT, GMFM-D, and GMFM-E. Figure 14(a) shows the percentage of all patients who were clinically responders to 6MWT, and the percentage of all patients who had a 6MWT baseline deficit at dose 8 and dose 20. Figure 14(b) shows the percentage of all patients who were clinically responders to GMFM-D, and the percentage of all patients who had a 6MWT baseline deficit at dose 8 and dose 20. Figure 14(c) shows the percentage of all patients who were clinically responders to GMFM-E, and the percentage of all patients who had a 6MWT baseline deficit at dose 8 and dose 20.

[0068] Figure 15 shows the site-specific polyethylene glycolation analysis of different batches of Co-rhARG1-PEG. Detailed Embodiments

[0069] Recombinant Human Arginase 1

[0070] Human arginase 1, identified as hArg1, is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine (L-Arg) to produce L-ornithine and urea. Arginase 1 is a trimer of three non-covalently bonded identical monomeric units. Monomeric arginase 1 has enzymatic activity but is not very stable. Replacing the natural manganese (Mn2+) with cobalt (Co2+) at the active site of arginase 1 enhances its catalytic activity at physiological pH. The method described herein for producing cobalt-substituted arginase 1 provides an enzyme of high purity and high activity. This method can also provide Co-arginase 1 (Co-rhARG1) as a separation intermediate in the manufacture of active pharmaceutical ingredients. In one or more embodiments, the active pharmaceutical ingredient is polyethylene glycolated Co-arginase 1 (Co-rhARG1-PEG). PEGylation of Co-arginase 1 significantly prolongs the cycling half-life. Similarly, although specific reference is made herein to rhARG1, the methods, formulations, and uses described herein can also be applied to rhARG2.

[0071] As used herein, the term “rhARG1” refers to recombinant human arginase 1 enzyme, such as a recombinant enzyme having at least 98% sequence identity with SEQ ID NO: 1.

[0072] As used herein, the terms “Co-rhARG1,” “Co-arginase 1 intermediate,” etc., refer to rhARG1 in which at least some of the natural manganese cofactors are substituted with cobalt. In one or more embodiments, Co-rhARG1 is Co-rhARG1-PEG.Separable intermediates in the production and / or purification process.

[0073] As used herein, the terms “Co-rhARG1-PEG”, “polyethylene glycolated Co-arginase 1”, etc., refer to Co-rhARG1 having one or more PEG units, such as those covalently linked to the enzyme at an N-terminal amino acid and / or at one or more free amines at one or more lysine residues.

[0074] The amount of Co-rhARG1-PEG active pharmaceutical ingredient can be expressed as the mass of the unpolyethylene glycolated enzyme. In one embodiment of the method, each mg (based on enzyme) of Co-rhARG1-PEG active pharmaceutical ingredient also contains approximately 1–2 mg of PEG, such as approximately 1.4 mg of PEG.

[0075] Figure 1(a) shows the amino acid sequence expressed in Escherichia coli. The hArg1 protein sequence was obtained from the NCBI database (UniProtKB: locus ARGI1_HUMAN, accession number P05089). Overlapping oligonucleotides were used in the PCR reaction to generate arginase 1 DNA, which was codon-optimized for expression in E. coli (Figure 1(b)). The 321-amino acid arginase 1 monomer expressed in E. coli lacked the N-terminal methionine found in the native human arginase 1 monomer. The calculated molecular weight of Co-arginase 1 was 34,721.6 Daltons (Table 1). The calculated molecular weight of the homotrimeric Co-arginase 1 was 104,164.8 Daltons. Arginase 1 has no disulfide bonds. Specification 8 / 39 pages 14 CN 121518448 A

[0076] Table 1: Structural information of exemplary Co-arginase 1 intermediates

[0077]

[0078] In one or more embodiments, the calculated molecular weight of the monomer Co-rhARG1-PEG was about 75-115 kDa. In one or more embodiments, the calculated molecular weight of the homotrimeric Co-rhARG1-PEG is about 224-344 kDa. In one or more embodiments, the average number of PEGs is about 8 to about 25 moles of PEG / mole of Co-arginase 1 monomer, such as about 8 to about 16 moles of PEG / mole of Co-arginase 1 monomer. Exemplary amounts of PEG include about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, and about 16 moles of PEG / mole of Co-arginase 1 monomer. In one or more embodiments, each PEG has an average molecular weight of about 1,000 to about 10,000 Daltons, such as about 1,000, about 2,000, about 3,000, and about...4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, or about 10,000 Daltons. In a particular embodiment, the average MW of the PEG is about 5,000 Daltons.

[0079] In one or more embodiments, Co-rh ARG 1-PEG contains polyethylene glycol arginase. Polyethylene glycol arginase has the following two chemical names:

[0080] a. α-(carboxymethyl)-ω-methoxy-poly(oxy-1,2-ethylenedimethyl)amide and arginase 1 [cobalt cofactor] (synthetic human) (1:10) trimer

[0081] b. Des-Met1-arginase-1 (liver-type arginase, EC 3.5.3.1) (Homo sapiens), in which manganese has been replaced by cobalt, and an average of 10 primary amines (N-terminal serine and N6-lysine) are amidated with [methoxy-poly(ethyleneoxy)]acetyl groups, a non-covalent homotrimer, produced in

[0082] Escherichia coli. The molecular formula of polyethylene glycol arginase is C1554H2492N416O453S6 [C3H4O2 (C2H4O)n]a monomer. The average molecular weight of the trimer polyethylene glycol arginase is 284 kDa. The CAS registration number of polyethylene glycol arginase is 1659310-95-8.

[0083] The potential PEGylation sites of polyethylene glycol arginase are as follows:

[0084]

[0085] Typically, the PEGylation reaction is carried out on Co-rhARG1. In some embodiments, rhARG1 can be PEGylated. In one or more embodiments, the amount of reactants, time, temperature and solution, and reactant treatment (such as mixing, addition rate, PEG treatment) are important for producing consistent PEGylated products. Typically, the PEGylation reaction on Co-rhARG1 is carried out in a reaction buffer at pH 8.4. In one or more embodiments, Co-rhARG1 is PEGylated in 0.1 M sodium phosphate buffer at pH 8.4. In one or more embodiments, the polyethylene glycolation reaction includes a reactant ratio, i.e., a ratio of PEG (g) to Co-rhARG1 (g), in the range of 4:1 to 1:1. In one or more embodiments, the polyethylene glycolation reaction includes a reactant ratio, i.e., a ratio of PEG (g) to Co-rhARG1 (g) of about 2.77:1. In one or more embodiments, by misting PEG and Co-rhARG1...The polyethylene glycol (PEG) reaction is carried out over a period of approximately 5 to 300 minutes, approximately 10 to 300 minutes, approximately 20 to 300 minutes, approximately 30 to 300 minutes, approximately 5 to 280 minutes, approximately 10 to 280 minutes, approximately 20 to 280 minutes, approximately 30 to 280 minutes, approximately 5 to 260 minutes, approximately 10 to 260 minutes, approximately 20 to 260 minutes, approximately 30 to 260 minutes, approximately 5 to 240 minutes, approximately 10 to 240 minutes, approximately 20 to 240 minutes, and approximately 30 to 240 minutes. In one or more embodiments, the PEG reaction is stopped by removing excess PEG and lowering the pH of the reaction buffer. In some embodiments, excess PEG is removed by filtration. In one or more embodiments, the pH is lowered by exchanging the reaction buffer with the storage buffer. In some embodiments, the storage buffer comprises 5 mM potassium phosphate, 50 mM NaCl, 1.5% w / v glycerol, and pH 7.4.

[0086] In one or more embodiments, Co-rhARG1-PEG is PEGylated at one or more of the amino acid residues K16, K32, K38, K40, K47, K67, K74, K82, L87, K88, K152, K154, K171, K222, K223, K312, and K321. In some embodiments, Co-rhARG1-PEG is PEGylated at at least the amino acid residues K16, K32, K38, K40, K47, K67, K74, K82, L87, K88, K152, K154, K171, K312, and K321. In some embodiments, Co-rhARG1-PEG is PEGylated at amino acid residues K222 and / or K223. In some embodiments, Co-rhARG1-PEG is not PEGylated at amino acid residues K222 and / or K223. In some embodiments, Co-rhARG1-PEG is not PEGylated at one or more of amino acid residues K3, K149, K190, K195, K29, K265, and K283. In some embodiments, Co-rhARG1-PEG is not PEGylated at amino acid residues K3, K149, K190, K195, K29, K265, and K283.

[0087] In one or more embodiments of Co-rhARG1-PEG, K16 is in the range of about 15% to about 60%.K32 is PEGylated in the range of about 35% to about 80% in one or more embodiments of Co-rhARG1-PEG. K38 is PEGylated in the range of about 20% to about 85% in one or more embodiments of Co-rhARG1-PEG. K40 is PEGylated in the range of about 10% to about 60% in one or more embodiments of Co-rhARG1-PEG. K47 is PEGylated in the range of about 10% to about 60% in one or more embodiments of Co-rhARG1-PEG. K67 is PEGylated in the range of about 40% to about 90% in one or more embodiments of Co-rhARG1-PEG. K74 is PEGylated in the range of about 30% to about 95% in one or more embodiments of Co-rhARG1-PEG. In one or more embodiments of Co-rhARG1-PEG, K82 is polyethylene glycol-modified in the range of about 30% to about 98%. In one or more embodiments of Co-rhARG1-PEG, K87 is polyethylene glycol-modified in the range of about 15% to about 65%. In one or more embodiments of Co-rhARG1-PEG, K88 is polyethylene glycol-modified in the range of about 25% to about 70%. In one or more embodiments of Co-rhARG1-PEG, K152 is polyethylene glycol-modified in the range of about 25% to about 85%. In one or more embodiments of Co-rhARG1-PEG, K154 is polyethylene glycol-modified in the range of about 15% to about 65%. In one or more embodiments of Co-rhARG1-PEG, K171 is polyethylene glycol-modified in the range of about 20% to about 75%. In one or more embodiments of Co-rhARG1-PEG, K222 is PEGylated in the range of 0% to about 30%. In one or more embodiments of Co-rhARG1-PEG, K223 is PEGylated in the range of 0% to about 35%. In one or more embodiments of Co-rhARG1-PEG, K312 is PEGylated in the range of 0% to about 45%. In one or more embodiments of Co-rhARG1-PEG, K321 is PEGylated in the range of 0% to about 45%.

[0088] The PEG-protein molar ratio is an attribute indicator of the degree of PEGylation. In one or more embodiments, it is about 1 to about 20.One mole of PEG has been used to PEGylate one mole of Co-rhARG1. Exemplary ranges for the PEG:Co-rhARG1 molar ratio include 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1. In some embodiments, the PEG:Co-rhARG molar ratio is in the range of about 7 mol / mol to about 15 mol / mol.

[0089] Free PEG is measured to demonstrate PEG scavenging and stability. In some embodiments, the free PEG concentration (μg) in the polyethylene glycolated Co-rhARG1 (mL) is less than or equal to 500 μg / mL, less than or equal to 400 μg / mL, less than or equal to 300 μg / mL, less than or equal to 200 µg / mL, less than or equal to 100 µg / mL, and less than or equal to 50 µg / mL.

[0090] Human arginase 1 catalyzes the fifth and final step of the urea cycle, namely the conversion of L-arginine to L-ornithine and urea. The polyethylene glycolated active pharmaceutical ingredient Co-rhARG1-PEG catalyzes the same reaction. The enzyme activity was assessed by measuring the conversion of L-arginine to L-ornithine at pH 7.4 and 37°C for a fixed reaction time. The conversion of the product was converted into a reaction rate and fitted to the Michaelis-Menten equation to determine Km and kcat.

[0091]

[0092] Vmax is the maximum reaction rate achieved at the saturated substrate concentration; Km is the Michaelis-Menten binding constant, used to measure the substrate concentration at which half the rate of Vmax is obtained. The enzyme turnover number kcat is calculated from Vmax / [E].

[0093] Specific activity is determined by dividing the reaction rate (in μmol / min) at 2 mM arginine by the enzyme concentration (in mg).

[0094] The KM and kcat values ​​of the Co-rhARG1-PEG active pharmaceutical ingredient measured in enzyme activity assays are typically 0.15–0.22 mM and approximately 200–300 / sec, respectively. After PEGylation of the Co-arginase 1 intermediate to form the active pharmaceutical ingredient, there is no significant change in enzyme activity compared to the unPEGylated intermediate. However, compared to the Co-arginase 1 intermediate, PEGylation significantly increased the cycling half-life of the Co-rhARG1-PEG drug product.

[0095] In one or more embodiments, the protein (e.g., Co-rhARG1 or Co-rhARG1-PEG) at pHAt pH 7.4, the protein exhibits a kcat / KM greater than 200 mM⁻¹ s⁻¹. In a particular embodiment, the protein exhibits a kcat / KM in the range of about 200 mM⁻¹ s⁻¹ to about 4,000 mM⁻¹ s⁻¹ at pH 7.4. In another embodiment, the protein exhibits a kcat / KM in the range of about 400 mM⁻¹ s⁻¹ to about 2,500 mM⁻¹ s⁻¹ at pH 7.4 and 37°C. In a particular embodiment, the present invention contemplates a protein comprising the amino acid sequence of human arginase 1 and a non-natural metal cofactor, wherein the protein exhibits a kcat / KM greater than 400 mM⁻¹ s⁻¹ at 37°C and pH 7.4. Exemplary kcat / KM values ​​include about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 800, about 900, about 1,000, about 1,100, about 1,200, about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, and about 4,000 mM⁻¹ s⁻¹, or any range between these values, at pH 7.4 and 37°C.

[0096] Specific activity is an indicator of the potency of a protein (e.g., Co-rhARG1 or Co-rhARG1-PEG). In one or more embodiments, the specific activity of Co-rhARG-PEG is in the range of about 200 U / mg to about 1000 U / mg. Exemplary ranges of specific activity include about 200 U / mg to about 1000 U / mg, about 300 U / mg to about 1000 U / mg, about 400 U / mg to about 1000 U / mg, about 200 U / mg to about 900 U / mg, about 300 U / mg to about 900 U / mg, about 400 U / mg to about 900 U / mg, about 200 U / mg to about 800 U / mg, about 300 U / mg to about 800 U / mg, about 400 U / mg to about 800 U / mg, about 200 U / mg to about 700 U / mg, about 300 U / mg to about 700 U / mg, and about 400 U / mg to about 700 U / mg.

[0097] In one or more embodiments, rhARG1, Co-rhARG1, or Co-rhARG1-PEG may have at least [a specific chemical relationship] with SEQ ID NO: 1.98%, 98.5%, 99%, or 99.5% identity. In one or more embodiments, rhARG1, Co-rhARG1, or Co-rhARG1-PEG may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more deletions, substitutions, and / or insertions relative to the amino acid sequence described by SEQ ID NO: 1. Various alignment algorithms and / or procedures can be used to calculate the identity between two sequences, including FASTA or BLAST, which are available on the website of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0098] In one or more embodiments, rhARG1, Co-rhARG1, or Co-rhARG1-PEG has at least one amino acid substitution at a position selected from H100, D123, H125, D127, D231, D233, D180, S229, and C302. In some embodiments, rhARG1, Co-rhARG1, or Co-rhARG1-PEG contains at least one amino acid substitution selected from the group consisting of D180S, S229C, S229G, C302F, C302I, E255Q, D180E, and S229A. In one or more embodiments, rhARG1, Co-rhARG1, or Co-rhARG1-PEG contains at least one amino acid substitution at C302.

[0099] Using the methods described herein, almost all manganese cofactors in arginase 1 can be replaced with cobalt. Changes in the cobalt cofactor resulted in a change in the Km of arginine at pH 7.4 from 2.8 mM to approximately 0.18 mM. In one or more embodiments, Co-rhARG1-PEG contains approximately 0.1 to approximately 2 μg Co / mg protein. Exemplary cobalt loadings include approximately 0.1, approximately 0.2, approximately 0.3, approximately 0.4, approximately 0.5, approximately 0.6, approximately 0.7, approximately 0.8, approximately 0.9, approximately 1, approximately 1.1, approximately 1.2, approximately 1.3, approximately 1.4, approximately 1.5, approximately 1.6, approximately 1.7, approximately 1.8, approximately 1.9, and approximately 2 μg Co / mg protein.

[0100] Free cobalt was measured to demonstrate cobalt scavenging and stability. In some embodiments, the free cobalt concentration is less than or equal to 0.10 μg / mL, less than or equal to 0.09 μg / mL, less than or equal to 0.08 μg / mL, less than or equal to 0.07 μg / mL, less than or equal to 0.06 µg / mL, less than or equal to 0.05 µg / mL, and less than or equal to 0.04 µg / mL.

[0101] Total cobalt affects the efficacy of proteins and is an indicator of cobalt binding, since the amount of free cobalt is relatively small. In some embodiments, the total cobalt concentration is from about 5 µg / mL to about 20 µg / mL, from about 6 µg / mL to about 20 µg / mL, from about 7 µg / mL to about 20 µg / mL, from about 8 µg / mL to about 20 µg / mL, from about 9 µg / mL to about 20 µg / mL, from about 5 µg / mL to about 19 µg / mL, from about 6 µg / mL to about 19 µg / mL, from about 7 µg / mL to about 19 µg / mL, from about 8 µg / mL to about 19 µg / mL, from about 9 µg / mL to about 19 µg / mL, from about 5 µg / mL to about 18 µg / mL, from about 6 µg / mL to about 18 µg / mL, from about 7 µg / mL to about 18 µg / mL, from about 8 µg / mL to about 18 µg / mL, from about 9 µg / mL to about 18 µg / mL, from about 5 µg / mL to about 17 µg / mL. The ranges from approximately 6 µg / mL to approximately 17 µg / mL, from approximately 7 µg / mL to approximately 17 µg / mL, from approximately 8 µg / mL to approximately 17 µg / mL, from approximately 9 µg / mL to approximately 17 µg / mL, from approximately 5 µg / mL to approximately 16 µg / mL, from approximately 6 µg / mL to approximately 16 µg / mL, from approximately 7 µg / mL to approximately 16 µg / mL, from approximately 8 µg / mL to approximately 16 µg / mL, from approximately 9 µg / mL to approximately 16 µg / mL, from approximately 5 µg / mL to approximately 15 µg / mL, from approximately 6 µg / mL to approximately 15 µg / mL, from approximately 7 µg / mL to approximately 15 µg / mL, from approximately 8 µg / mL to approximately 15 µg / mL, and from approximately 9 µg / mL to approximately 15 µg / mL.

[0102] In various embodiments, Co-rhARG1-PEG contains less than about 1 μg Mn / mg protein, such as less than about 1, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.15, about 0.1, about 0.09, about 0.08, about 0.07, about 0.06, about 0.05, about 0.04, about 0.03, about 0.02, or about 0.01 μg Mn / mg protein. In a particular embodiment, the Co-rhARG1-PEG active pharmaceutical ingredient contains about 2 μg Co / mg protein and about 0.05 μg Mn / mg protein. (See page 12 / 39 of the specification, 18 CN)121518448 A μg Mn / mg protein.

[0103] In various embodiments, Co-rhARG1-PEG contains less than about 1 μg Fe / mg protein, such as less than about 1, about 0.9, about 0.8, about 0.7, about 0.6, about 0.5, about 0.4, about 0.3, about 0.2, about 0.15, about 0.1, about 0.09, about 0.08, about 0.07, about 0.06, about 0.05, about 0.04, about 0.03, about 0.02, or about 0.01 μg Fe / mg protein.

[0104] Production and purification of rhARG1, Co-rhARG1, and PEGrhARG1

[0105] An overview of exemplary upstream and downstream production methods is shown in Figures 2 and 3.

[0106] Shake Flask Expansion

[0107] The purpose of shake flask expansion / fermentation is to generate inoculum for use in the production fermenter. Shake flask expansion produces cell clusters for inoculation of the production reactor and additional cell clusters for analytical purposes. A representative overview of the arginase 1 fermentation process is shown in Figure 2.

[0108] Aliquots of the inoculation medium were introduced into one 500 mL flask (primary flask) and six 3 L disposable flasks (secondary flasks). The flasks were autoclaved, and the sterilized additives were transferred to each flask. The primary medium was preheated to a treatment temperature of 37°C before inoculation. The secondary flasks were preheated to a treatment temperature of 37°C before secondary inoculation.

[0109] A bottle of working E. coli cell bank (WCB) expressing arginase 1 was removed from the freezer and thawed. The target volume of thawed cells (approximately 1.1 mL) was aseptically added to the primary flask, and the flask was incubated at 37°C with stirring. Starting a few hours after inoculation, samples were taken from the flasks hourly to track cell growth by optical density (OD600) at 600 nm. Once the target OD600 in the primary flask reached ≥ 1.0, a target volume (15 mL) of the primary culture was aseptically transferred to each secondary flask. The secondary flasks were incubated at 37°C with stirring. Starting 4 hours after inoculation, samples were taken from one secondary flask hourly, increasing to once every 30 minutes once the OD600 reached ≥ 1.5. When the specified density of ≥ 2.0 OD600 was measured, the remaining secondary flasks were sampled. If the average OD600 of all secondary flasks met the specified transfer criteria, the flasks were combined and the inoculum was transferred to the production fermenter. A representative overview of the arginase 1 fermentation process is shown in Figure 2.

[0110] Production Fermentation

[0111] The purpose of the production fermentation was to scale up the shake flask culture and induce arginase 1.Production. Production fermentation can produce large quantities of arginase 1. After establishing cell clusters during the shake-flask expansion phase, the fermentation process produces arginase 1 (in E. coli) as a soluble protein. In one embodiment, a 1500 L fermenter contains an initial batch medium including sterile additives prior to inoculation. After inoculation, the fermenter input includes nutrient feed, antifoaming solution, and the addition of acid or alkali to maintain the pH of the culture. A secondary container holds the nutrient feed medium. An automated control strategy maintains critical parameters for consistent cell growth, including dissolved oxygen, jet rate, agitation rate, pH, pressure, and temperature. Arginase 1 expression is induced by the addition of IPTG (isopropyl β-D-1-thiogalactoside) and harvested approximately 18 hours later. At the end of production, fermenter performance is evaluated by monitoring cell density, solids percentage, and the proportion of soluble arginase 1.

[0112] In a preferred embodiment, the fermentation medium is prepared directly in the production fermenter. Purified water is added to the fermentation medium to the desired weight prior to in-situ sterilization (SIP). After the culture medium cooled, the sterile additives kanamycin, glucose, and potassium phosphate were sterilely filtered into the production fermenter. If necessary, the sterile culture medium was mixed with purified water to the specified pre-inoculation weight using a 0.2 µm sterile filter. The fermentation medium was titrated to a controlled pH with alkali (ammonium hydroxide).

[0113] The combined inoculum was aseptically inoculated into the production fermenter at 37°C using pressure-assisted transfer. Fermentation broth samples were collected and measured at a fixed frequency according to the specification (pages 13 / 39, CN 121518448 A) for OD600 analysis from inoculation time to fermentation cooling. Glucose samples were collected at fixed intervals starting 3 hours after inoculation, with the frequency increased at 9 hours after inoculation. Antifoaming solution was added as needed during fermentation to avoid excessive foaming of the culture. Dissolved oxygen was controlled by a stirring cascade, with oxygen injected as needed. Acid and alkali inputs were used to maintain the pH of the culture. The growth medium is preferably maintained at 36-38°C and pH 7.0-7.4, with stirring and aeration.

[0114] The nutrient feed consists of yeast extract, Martone B-1, L-cysteine ​​hydrochloride, and glycerol. Feeding begins when the glucose concentration is less than 10 g / L (12-14 hours after inoculation) and continues at a fixed rate until the end of production. Expression is triggered by the addition of IPTG. Induction lasts for 18 hours. After the fermentation process is completed, the mixture is cooled in preparation for the harvest operation. The production fermenter can produce a soluble arginase 1 titer of about 6 g / L. An overview of the production fermentation is shown in Figure 3.

[0115] Harvesting Operation

[0116] The harvest operation captures the soluble arginase 1.The cells are broken up / lysed, and cell debris is removed from the lysate by centrifugation and / or filtration. The recovered cytoplasm can be frozen or cryopreserved for long-term storage. The harvesting operation can be performed by collecting cells by centrifugation, lysing them twice by homogenizer or by breaking them under pressure (French press), centrifuging a second time, and membrane filtration before the first chromatographic step.

[0117] In a preferred embodiment, whole cells are separated from the fermentation medium using a disc centrifuge. The resulting cytoplasm is resuspended in 25 mM HEPES at pH 7.6 and then treated twice by homogenizer. Alternatively, a pH of 25 mM HEPES in the range of pH 7.2–7.6 can be used. The lysed material is clarified using a centrifuge to remove cell debris and then membrane filtered through a 0.2 μm filter. In a preferred embodiment, the harvesting step is performed at a target temperature of ≤ 15°C.

[0118] In another embodiment, cell disruption is performed using high pressure. The cell plasma is transferred to a homogenizer at a controlled rate, and the homogenized effluent is passed through a heat exchanger to reduce the temperature rise that occurs during pressure homogenization. Frozen cells undergo two homogenizations. The combined product of the first lysis is transferred from the collection container back to the feed container. The holding time between the two treatments is minimized to reduce potential microbial growth.

[0119] The lysed material is clarified by centrifugation to remove cell debris from the soluble components of the lysate. The lysate is transferred to a disc-type batch discharge centrifuge at a controlled rate. The clarified lysate is collected for further processing.

[0120] The clarified lysate is filtered, such as using a filter of about 0.2 µm. A process transfer filter can also be used for microbial control during process operation. For this purpose, the filter can be a 0.5 µm or 0.2 µm filter. This step also removes small particles from the clarified material that may not have been separated during the clarification operation. Before use, the filter is thoroughly rinsed with purified water and equilibrated with 25 mM HEPES buffer, pH 7.6. A pre-filter can be installed before each downstream process step to mitigate potential biological burden.

[0121] Purification of rhARG1, Co-rhARG1, and Co-rhARG1-PEG

[0122] Regardless of the method used to culture cells expressing rhARG1 (e.g., the fermentation process described above), the purification methods described herein can be used to capture rhARG1 and further purify the enzyme. Purification methods may include optional steps such as loading cobalt to produce Co-rhARG1 and / or reacting with a polyethylene glycol reaction product to provide Co-rhARG1-PEG.

[0123] Various embodiments of the purification process involve using a cation exchange (CEX) column to capture rhARG1. In one or moreIn several embodiments, the CEX column is the first column (“Column 1”) in a system having multiple chromatographic columns. The protein product eluted from Column 1 is the “first protein product”.

[0124] In one or more embodiments, Column 1 binds rhARG1 using cation exchange chromatography at a pH in the range of about 7 to about 8, such as about 7.6. In one or more embodiments, rhARG1 binds at a salt-free or low-salt concentration. In one or more embodiments, rhARG1 is eluted with a buffer having a high salt (e.g., NaCl) concentration, such as up to about 0.5 M NaCl. Exemplary salt concentrations include about 0.01, about 0.02, about 0.03, about 0.04, about 0.05, 0.1, about 0.2, about 0.3, about 0.4, and about 0.5 M NaCl.

[0125] In various embodiments, salt gradients are used to separate different charge variants of rhARG1. Exemplary salt gradients are about 0 to about 0.5 M NaCl, about 0 to about 0.4 M NaCl, about 0 to about 0.3 M NaCl, about 0 to about 0.2 M NaCl, or about 0 to about 0.1 M NaCl.

[0126] In one or more embodiments, the method further includes loading a first protein product (optionally after cobalt substitution) onto an anion exchange (AEX) column (“Column 2”) and collecting the flow to provide a second protein product (“Second Protein Product”). In another aspect of the method, the method further includes loading the second protein product onto a third column for capturing arginase 1 and then eluting it to provide a third protein product (“Third Protein Product”). In some embodiments, the third column (“Column 3”) may be a size exclusion chromatography (SEC) column or a multimode chromatography (MMC) column.

[0127] Various embodiments provide rhARG1 loaded with Co to replace the Mn cofactor. In one or more embodiments, Co2+ salts such as CoCl2 are used for Co loading. The incubation time is temperature-dependent, so lower cobalt substitution temperatures require longer incubation times, and higher cobalt substitution temperatures do not require longer incubation times. Cobalt loading temperatures can be as low as 1°C or above 50°C, while the corresponding incubation times can be as long as 8 hours or less than 10 minutes.

[0128] Various embodiments provide rhARG1 or Co-rhARG1 reacted with a polyethylene glycolation reactant such as methoxyPEG succinimide carboxymethyl ester (MW 5000). Compared to enzymes, the polyethylene glycolation reactant is typically reacted at a molar concentration of 10-40.The amount of arginine is provided in excess. The incubation time can range from 0.5 to 4 hours. The pH during PEGylation can be from about 8 to about 9, such as a pH of about 8.4.

[0129] In one or more embodiments, the purified PEGylated protein rhARG1 or Co-rhARG1 comprises arginase I monomer, glucosylated arginase I, phosphogluconate arginase I, 2x glucosylated arginase I, glucosylated + phosphogluconate arginase I, and 2x phosphogluconate arginase I.

[0130] In some embodiments, the purified PEGylated protein rhARG1 or Co-rhARG1 comprises at least 70% arginase I monomer. Exemplary amounts include at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% arginase I monomer. In some embodiments, the purified PEGylated protein rhARG1 or Co-rhARG1 contains less than 10% glucosylated arginase I. Exemplary amounts include about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, or about 9% glucosylated arginase I, or any range between these values. In some embodiments, the purified PEGylated protein rhARG1 or Co-rhARG1 contains less than 10% phosphoglucono-γ arginase I. Exemplary amounts include about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, or about 9% phosphoglucono-γ arginase I. In some embodiments, the purified PEGylated protein rhARG1 or Co-rhARG1 contains at least 70% arginase I monomer, less than 10% glucosylated arginase I, and less than 10% phosphoglucosylated arginase I.

[0131] Due to the level of heterogeneity in the PEGylated trimer, imaging capillary isoelectric focusing (iCIEF) provides a measurement of the consistency of the PEGylated protein. In one or more embodiments, iCIEF analysis of the purified PEGylated protein rhARG1 or Co-rhARG1 contains nine distinct peaks, namely peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak 7, peak 8, and peak 9, each corresponding to nine substances with different charges, namely substance 1, substance 2, substance 3, substance 4, substance 5, substance 6, substance 7, substance 8, and substance 9. The area under the curve for each peak corresponds to page 15 / 39 of the specification sheet for that specific substance, 21 CN 121518448.The proportion of A. In one or more embodiments, certain peaks may be combined together for related substances, such as peak 1+2 or peak 3+4.

[0132] In some embodiments, the purified polyethylene glycol-modified protein rhARG1 or Co-rhARG1 contains the area under the curve of peak 1 in proportions less than about 30%, less than about 25%, less than about 20%, less than about 15%, and less than about 10%.

[0133] In some embodiments, the purified polyethylene glycol-modified protein rhARG1 or Co-rhARG1 contains the area under the curve of peak 2 in proportions less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, and less than about 10%.

[0134] In some embodiments, the purified polyethylene glycol-modified protein rhARG1 or Co-rhARG1 contains the area under the curve of peak 1+2 in proportions of less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, and less than about 10%.

[0135] In some embodiments, the purified polyethylene glycol-modified protein rhARG1 or Co-rhARG1 contains the area under the curve of peak 3+4 in the range of about 2% to about 40%, about 2% to about 35%, about 2% to about 30%, about 2% to about 25%, about 4% to about 40%, about 4% to about 35%, about 4% to about 30%, about 4% to about 25%, about 6% to about 40%, about 6% to about 35%, about 6% to about 30%, about 6% to about 25%, about 8% to about 40%, about 8% to about 35%, about 8% to about 30%, about 8% to about 25%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, and about 10% to about 25%.

[0136] In some embodiments, the purified polyethylene glycol-modified protein rhARG1 or Co-rhARG1 contains the area under the curve of peak 5 in the range of about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, and about 15% to about 25%.

[0137] In some embodiments, the purified polyethylene glycol-modified protein rhARG1 orCo-rhARG1 contains the area under the curve of peak 6 in the range of about 2% to about 35%, about 2% to about 30%, about 2% to about 25%, about 2% to about 20%, about 4% to about 35%, about 4% to about 30%, about 4% to about 25%, about 4% to about 20%, about 6% to about 35%, about 6% to about 30%, about 6% to about 25%, about 6% to about 20%, about 8% to about 35%, about 8% to about 30%, about 8% to about 25%, about 8% to about 20%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, and about 10% to about 20%.

[0138] In some embodiments, the purified polyethylene glycol-modified protein rhARG1 or Co-rhARG1 contains an area under the curve (AUC) of peak 7 in a proportion less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, and less than about 10%.

[0139] In some embodiments, the purified polyethylene glycol-modified protein rhARG1 or Co-rhARG1 contains an AUC of peak 8 in a proportion less than about 25%, less than about 20%, less than about 15%, less than about 10%, and less than about 5%.

[0140] In some embodiments, the purified polyethylene glycol-modified protein rhARG1 or Co-rhARG1 contains an AUC of peak 9 in a proportion less than about 18%, less than about 16%, less than about 14%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, and less than about 4%.

[0141] Administration of rhARG1, Co-rhARG1 and Co-rhARG1-PEG

[0142] rhARG1, Co-rhARG1 and Co-rhARG1-PEG (and compositions comprising them) as described herein can be administered by any suitable route, including intravenous, intrathecal, subcutaneous, intramuscular, intratumoral and / or intraperitoneal. In one or more embodiments, rhARG1, Co-rhARG1 and Co-rhARG1-PEG (or compositions comprising them) are administered intravenously (IV) or subcutaneously (SC). Specification 16 / 39 pages 22 CN 121518448 A

[0143] Compositions containing rhARG1, Co-rhARG1 and Co-rhARG1-PEG can be provided in formulation form with physiologically tolerable liquid, gel or solid carriers, diluents and excipients. Such compositions are generally prepared as liquidsSolutions or suspensions, as injectables. Suitable diluents and excipients are, for example, water, saline, glucose, glycerol, etc., and combinations thereof. In addition, if desired, the composition may contain small amounts of excipients, such as wetting agents or emulsifiers, stabilizers or pH buffers.

[0144] Exemplary methods and instructions for administering rhARG1, Co-rhARG1, and Co-rhARG1-PEG (e.g., polyethylene glycol arginase) are provided below. Although the following description is specific to polyethylene glycol arginase, these methods and instructions are also applicable to other recombinant arginase 1 and 2 enzymes.

[0145] Recommended intravenous administration regimen:

[0146] Obtain baseline plasma arginine concentrations before initiating treatment. The initial recommended dose of polyethylene glycol arginase for ARG1-D patients is 0.10 mg / kg, administered once weekly as a single intravenous infusion. If an initial dose of 0.10 mg / kg fails to reduce plasma arginine to ≤150 μmol / L, the dose may be adjusted to a maximum of 0.20 mg / kg, once weekly. If plasma arginine levels drop below 50 μmol / L during treatment, dose reduction should be considered. Subcutaneous administration of PEGylated arginase may be considered for ARG1-D patients after 5 or more IV doses, with continued periodic monitoring of plasma arginine levels.

[0147] Recommended subcutaneous administration regimen:

[0148] When transitioning from intravenous to subcutaneous administration of PEGylated arginase, the first subcutaneous dose should be administered, not the planned next intravenous dose. The initial subcutaneous dose should be the same as the mg / kg dose of the last IV dose. The subcutaneous dose may be adjusted as clinically indicated to ensure that plasma arginine levels remain within the range of 50 to 150 μmol / L.

[0149] Blood Arginine Monitoring:

[0150] Plasma arginine monitoring should be performed after initiation of PEGylated arginase treatment until the patient's plasma arginine level is within the target range of 50 to 150 µmol / L. Thereafter, regular monitoring of plasma arginine is recommended to assess blood arginine control. Additional plasma arginine monitoring may be required when switching to subcutaneous administration or changing diet.

[0151] Preparation and Administration Instructions

[0152] PEGylated arginase is provided as a frozen liquid formulation in 10 mL single-use glass vials containing 5 mL of PEGylated arginase at a concentration of 1 mg / mL or 5 mg / mL. Each single-use glass vial of PEGylated arginase is intended for use as a single intravenous or subcutaneous injection. Visual inspection of the PEGylated arginase prior to administration is required.Check for particulate matter and discoloration of PEG arginase. PEG arginase is a colorless to pale yellow or pale pink solution. If discolored, cloudy, or with particulate matter in the vial, discard it. Remove the cap from the vial. Disinfect the vial stopper with an alcohol swab. Using a sterile syringe with an 18G needle, draw an appropriate volume of the drug from the vial. If more than one vial is needed, draw the solution from each vial using a separate needle. Calculate the amount of solution to be drawn from the vial for the infusion pump. After drawing an appropriate volume of the drug into the syringe, draw saline using a separate needle to reach a total volume of 40 mL. Calculate the required amount of drug as follows:

[0153]

[0154] Administer PEG arginase via intravenous infusion over 30 minutes using an infusion pump. Instructions for Use, Pages 17 / 39, 23 CN 121518448 A

[0155] Table 2: Weight-based dosing for once-weekly administration of 0.1 mg / kg

[0156]

[0157] In one or more embodiments, the volume for subcutaneous injection has a maximum volume, such as a maximum of 2 mL per injection for adult patients and / or a maximum volume of 1 mL per injection for pediatric patients. If the calculated subcutaneous administration volume is greater than the maximum volume, a higher vial concentration (e.g., 5 mg / mL instead of 1 mg / mL) may be used and / or the volume may be divided into multiple smaller injections (e.g., dividing a 4 mL injection into two 2 mL injections).

[0158] Dosage Form and Strength

[0159] Polyethylene glycol arginase injection is a colorless to slightly yellow or slightly pink solution available as follows, in 10 mL vials:

[0160] a. Injection: 5 mL, 1.0 mg / mL

[0161] b. Injection: 5 mL, 5.0 mg / mL

[0162] Warnings and Precautions Instructions for Use 18 / 39 pages 24 CN 121518448 A

[0163] Hypersensitivity reactions may occur with the use of polyethylene glycol arginase. Monitor all patients for signs and symptoms of acute anaphylactic reactions (e.g., urticaria, pruritus, erythema, hypotension, tachycardia) during and after polyethylene glycol arginase infusion. In the event of a severe hypersensitivity reaction, immediately slow or discontinue the administration of polyethylene glycol arginase and administer appropriate medical care. Consider preoperative administration of a non-sedating antihistamine to the patient prior to administration. In cases where corticosteroids are required, they should be used with caution as they may cause hyperammonemia.

[0164] Pregnancy: Pregnancy Category B

[0165] Reproductive studies have been conducted in mice and rats at doses up to 100 mg / kg. There is no evidence that polymorphism...Glycol arginase can harm the fetus. However, there are no adequate and well-controlled studies in pregnant women. Since animal reproductive studies are not always predictive of human responses, PEGylated arginase should only be used during pregnancy when clearly needed.

[0166] Lactating Women

[0167] It is unknown whether PEGylated arginase is present in human milk. The developmental and health benefits of breastfeeding should be considered in conjunction with the mother's clinical need for PEGylated arginase and any potential adverse effects of the drug on breastfed infants.

[0168] Description

[0169] PEGylated arginase is a cobalt-substituted recombinant human arginase I enzyme covalently conjugated with monomethoxy polyethylene glycol (mPEG) and functions by catalyzing the same reaction as arginase 1, converting arginine to ornithine and urea. Human arginase 1 is a binuclear manganese metalloenzyme. To produce PEGylated arginase, the manganese cofactor is replaced by cobalt to produce Co-arginase I. Replacing natural manganese (Mn+2) with cobalt (Co+2) at the active site of arginase I enhances stability and catalytic activity at physiological pH. Polyethylene glycolization prolongs the cycling half-life. The average molecular weight of polyethylene glycol arginase is approximately 284 kDa. The specific activity range of polyethylene glycol arginase is approximately 320–600 units per mg of protein. One unit of activity is defined as the amount of enzyme required to convert 1 micromolar arginine to ornithine per minute at 37°C.

[0170] Polyethylene glycol arginase is intended for intravenous or subcutaneous infusion and is provided as a sterile, clear, colorless to pale yellow or pale pink solution, prepared at pH 7.4 in a buffer containing 50 mM sodium chloride, 5 mM potassium phosphate, and 1.5% w / v glycerol, at a concentration of 1 mg / mL and 5 mg / mL. It is provided as a preservative-free, sterile solution in single-use clear glass vials. Each vial of 1 mg / mL polyethylene glycol arginase finished product contains 5 mL of finished product (5 mg polyethylene glycol arginase per vial). Each vial of 5 mg / mL polyethylene glycol arginase finished product contains 5 mL of finished product (25 mg polyethylene glycol arginase per vial). The vials are sealed with coated rubber stoppers and aluminum flip caps, stored frozen at ≤ -60°C, and thawed before use.

[0171] Pharmacodynamics

[0172] In adult and pediatric patients with arginase 1 deficiency, treatment with polyethylene glycol arginase resulted in a decrease in blood arginine concentration from baseline to the normal blood arginine range of 40 to 115 μmol / L. Maximum inhibition of L-arginine was observed approximately 8 hours after administration, decreasing in a dose-dependent manner, and maximal inhibition was observed 168 hours after administration.The levels returned to pre-dose levels. A strong correlation was observed between polyethylene glycol arginase and arginine, with immediate inhibition of arginine following IV administration and a maximum reduction in arginine concentration within 24 hours of administration.

[0173] Pharmacokinetics

[0174] Pharmacokinetic samples were collected in 14 subjects over the entire dosing interval from 0 to 168 hours following IV administration to characterize the relationship between polyethylene glycol arginase pharmacokinetics and arginine. Throughout the dose range (0.015 mg / kg to 0.2 mg / kg), polyethylene glycol arginase exposure, as measured by Cmax and AUC0-168, increased substantially proportionally with the dose, with a 13-fold increase in dose resulting in a 14-fold increase in Cmax and AUC0-168. No accumulation of polyethylene glycol arginase was observed following a weekly IV dosing regimen, with a T1 / 2 of approximately 30 hours across the entire dose range and low to moderate inter-subject variability (13% to 46% CV) in the exposure metric.

[0175] Animal Toxicology and / or Pharmacology

[0176] The pharmacological effects of polyethylene glycol arginase on arginine levels were evaluated in neonatal transgenic mouse models of arginase I and adult mouse models of tamoxifen-induced arginase deficiency. These models simulate human disease in which there is a significant excess of circulating arginine and arginine catabolites; however, unlike humans with arginase I deficiency, these animals develop severe and often fatal hyperammonemia. Pharmacological effects were also evaluated in a rat model of arginine-induced hyperarginemia. PEGL arginase reduces plasma arginine levels in a dose-dependent manner.

[0177] The potential toxicity and TK of PEGylated arginine enzyme were assessed in juvenile rats at day 21 postnatal day (equivalent to 2-year-old humans) by weekly intravenous bolus injections of 0.1, 0.3, and 1.0 mg / kg for 6 months, followed by a 6-week recovery period. PEGylated arginine enzyme was well tolerated, with no test-related mortality or significant effects observed on food consumption, coagulation, urinalysis, ophthalmoscopy, sexual maturation, growth hormone analysis, bone marrow analysis, functional observation group (FOB) assessment, and neurobehavioral tests (auditory startle habituation, motor activity, or Morris water maze). No macroscopic findings related to PEGylated arginine enzyme were observed at the end of the 6-month period and at the end of the 6-week recovery interval. At 0.3 and 1.0 mg / kg, adverse microscopic changes were limited to the testes and epididymis and were associated with reduced weight of male reproductive organs and adverse sperm analysis results. At 1.0 mg / kg...Adverse effects on sperm analysis were observed at 0.3 mg / kg, including decreased sperm motility, reduced sperm count in the epididymal tail, decreased sperm concentration, and an increased percentage of abnormal sperm. These observations were considered to be effects directly related to treatment and were associated with microscopic changes in microtubule degeneration in the testes at 0.3 mg / kg and 1.0 mg / kg. These changes were generally reversible after a 6-week recovery period in the control and 1.0 mg / kg groups, except for the increased percentage of abnormal sperm and sperm count. The partial reversibility after 6 weeks was not unexpected, as the normal sperm development cycle is approximately 9 weeks or longer than the 6-week recovery period.

[0178] Importantly, no significant PEGylation effect was observed in histopathology. Toxicokinetic data indicated that PEG arginase exposure was maintained throughout the study. In summary, the NOAEL for women was 1.0 mg / kg. In males, the NOAEL was 0.1 mg / kg based on microscopic changes in the testes at 0.3 mg / kg and 1.0 mg / kg.

[0179] Cynomolgus monkeys were given intravenous bolus injections weekly at doses of 0.1, 0.3, and 1.0 mg / kg for 13 weeks, followed by a 4-week recovery period, after which the potential toxicity and TK of PEGylated arginase were assessed. Clinical signs observed at 1.0 mg / kg included weight loss, thinning hair (generalized), dry / discolored skin (generalized), tremors, loss of appetite, watery stools, decreased activity, ataxia, muscle atrophy, and / or an untidy / kyphotic appearance. No treatment-related effects were found in clinicopathological parameters (coagulation, growth hormone, and urinalysis), ECG and ophthalmological examination, respiratory rate, and blood pressure assessment.

[0180] How to provide / store and handle

[0181] PEGylated arginase is provided as an injectable solution.

[0182] Polyethylene glycol arginase is supplied frozen (≤ -60°C). Diluted polyethylene glycol arginase should be used immediately. If immediate use is not possible, diluted polyethylene glycol arginase can be stored at 2°C to 8°C (36°F to 46°F) for up to 8 hours during administration.

[0183] Example Specification 20 / 39 pages 26 CN 121518448 A

[0184] Before describing several exemplary embodiments of this disclosure, it should be understood that this disclosure is not limited to the details of the construction or process steps set forth in the following detailed description. This disclosure can be used in other embodiments and can be practiced or performed in various ways.

[0185] In the following experimental disclosure, the following abbreviations are used: eq (equivalent); M (molar concentration); µM (micromolar concentration); mM(millimol concentration); N (normal); mol (moles); mmol (millimols); µmol (micromoles); nmol (nanomoles) g (grams); mg (milligrams); µg (micrograms); L (liters); ml (milliliters); µl (microliters); cm (centimeters); mm (millimeters); µm (micrometers); nm (nanometers); MW (molecular weight); PBS (phosphate-buffered saline); min (minutes).

[0186] Example 1: Cation exchange column chromatography (column 1)

[0187] In a preferred embodiment, arginase 1 is captured on a cation exchange column (CEX) to reduce product-related impurities and process-related impurities such as host cell proteins (HCP), DNA, and endotoxins (see Figure 3 for an overview of the purification process). In a particular embodiment, the first column (column 1) chromatographic step in the arginase 1 purification process uses SP Sepharose FF resin and an inlet heat exchanger. Column 1 was used to bind arginase 1 at pH 7.6 in the absence of salt using cation exchange chromatography and eluted with a buffer solution of increasing salt concentration (Fig. 4(a)). In one embodiment, the salt was NaCl, and elution of Column 1 was performed at room temperature with 25 mM HEPES, 0.1 M NaCl, pH 7.2–7.6. However, alternative embodiments are possible, such as applying a NaCl gradient to Column 1.

[0188] Fig. 4(a) shows a representative purification of arginase 1 on Column 1. Approximately three liters of clear E. coli lysate were loaded onto a cation exchange column. The high absorbance at 280 nm indicates that a large amount of protein was not bound to the column but was detected in the flow-through. The column was then washed with approximately two liters of column wash solution. The fraction rich in arginase 1 (final peak) was then eluted with 0.1 M NaCl by detection at 280 nm.

[0189] Example 2: Cobalt Substitution

[0190] In a preferred embodiment, the natural manganese coenzyme of arginase 1 is replaced by cobalt. During cobalt substitution (also known as cobalt loading), one or both of the two manganese ions normally present in arginase 1 are replaced by cobalt ions. The cobalt substitution step can be performed at a variety of temperatures and a variety of cobalt concentrations (see Table 2). The incubation time for cobalt substitution can be as short as 10 minutes and carried out at 50°C or above. Conversely, the cobalt loading temperature can be as low as 1°C or 5°C and carried out for more than 8 hours. Furthermore, the higher the proportion of cobalt loaded into arginase 1, the higher the specific activity.

[0191] Arginase 1 eluted from column 1 (also known as column 1 confluent) can be maintained at room temperature for the cobalt substitution step. In one embodiment, this is achieved by using a cobalt chloride stock solution (0.5 M CoCl2).The stock solution was diluted 50-fold by adding it to the column 1 consumable at a prescribed rate, resulting in a final cobalt chloride concentration of 10 mM. The cobalt-substituted reactants were then mixed at 20°C for two hours. In another embodiment, arginase 1 cobalt loading was carried out at room temperature in a 10 mM CoCl2 solution for approximately 2 to approximately 8 hours.

[0192] An overview of the cobalt loading steps is shown in Table 3.

[0193] Table 3 Specification 21 / 39 pages 27 CN 121518448 A

[0194]

[0195] Example 3: Ultrafiltration / Distillation 1 (UF / DF 1)

[0196] UF / DF 1 removes free cobalt ions and exchanges Co-arginase 1 into the solution, preparing for anion exchange chromatography. The UF / DF 1 step uses a membrane with a molecular weight cutoff of 30 kDa. An important function of this step is to reduce the level of free cobalt and perform buffer exchange on the Co-arginase 1 consumable before anion exchange chromatography. The membrane was sterilized with a cleaning solution (0.5 N NaOH) and rinsed with water. A standardized permeability test (NWP) was performed, and the membrane was then equilibrated before use in production. Once the UF / DF system was equilibrated, the Co-arginase 1 confluence was percolated three times the percolation volume with 25 mM HEPES, 0.1 M NaCl, pH 7.6, and then four times the percolation volume with 50 mM Tris, pH 8.4. After percolation, the confluence was recycled from the system using twice the system retention volume of 50 mM Tris, pH 8.4.

[0197] The UF / DF1 membrane was cleaned by rinsing with 2 M NaCl, followed by a denaturing cleaning step with 0.5 N NaOH, and then recirculated for 30 minutes. The system was rinsed with purified water and the NWP was tested to assess the effectiveness of the cleaning procedure. The membrane could be stored in 0.1 N NaOH.

[0198] In another embodiment, the first buffer exchange is to 25 mM HEPES, 0.1 M NaCl, pH 7.2–7.6, and the second exchange is to 50 mM Tris, pH 8.1–8.5.

[0199] Example 4: Anion exchange column chromatography (Column 2)

[0200] A preferred embodiment of arginase 1 purification uses another column (“Column 2”) as anion exchange column chromatography. One embodiment of Column 2 is Q Sepharose FF resin. One function of this Column 2 step is to reduce process-related impurities in the UF / DF1 conjugate, such as host cell DNA and endotoxins. Column 2 binds these impurities, while Co-arginase 1...The flow-through is collected in the column effluent during the loading and washing steps. In one embodiment, anion exchange flow-through chromatography of column 2 is performed using Q Sepharose FF, and up to 40 g protein / L resin is loaded onto the column using buffer 50 mM Tris, pH 8.1–8.5.

[0201] In another embodiment of the method, a first protein product is loaded onto an anion exchange column to capture impurities while recovering arginase 1 in the flow-through. Figure 4(b) is a representative chromatogram of arginase 1 purified on an anion exchange column (column 2). As can be seen from the absorbance at 280 nm, a large amount of protein is detected in the flow-through. Impurities are captured on column 2 and are not eluted into the column 2 merge (also known as the second protein product), which further enriches arginase 1 in one step.

[0202] Example 5: Capto Multimode Column Chromatography (Column 3)

[0203] In a preferred embodiment, the arginase purification process uses a third column (Column 3). In one embodiment, Column 3 is a Capto Multimode Chromatography (MMC) column or alternatively a size exclusion column. In the embodiment using MMC, arginase 1 is captured on the column, while process-related impurities such as host cell proteins (HCP), DNA, and endotoxins are washed away in the flow. In this embodiment, Co-arginase 1 can be captured by the column at pH 8.4 in the absence of salt, and then eluted with a buffer with increased salt concentration. A representative example of a Capto Multimode Cation Exchange Column is shown in Figure 4(c).

[0204] In one embodiment, MMC chromatography (Column 3) uses approximately 15 column volumes to load up to 30 g protein / L resin and elutes with a high-salt step using 50 mM Tris, 250 mM NaCl, pH 8.1–8.5. In several embodiments, flow from the anion exchange column (column 2) was loaded onto a Capto MMC column at pH 8.4, washed, and then eluted with 50 mM tromethamine and 250 mM sodium chloride to remove the bound Co-arginase 1.

[0205] Example 6: Ultrafiltration / Percolation 2 (UF / DF 2)

[0206] UF / DF 2 concentrates arginase 1 and exchanges the protein into a pre-polyethylene glycolated intermediate. The UF / DF 2 step uses a membrane with a molecular weight cutoff of 30 kDa. An important function of this step is to allow the unpolyethylene glycolated Co-arginase 1 intermediate to pass through column 3 before polyethylene glycolation (or before additional filtration and storage).The confluent is buffer exchanged. The membrane is sterilized with a cleaning solution (0.5 N NaOH) and rinsed with water. Once the UF / DF system reaches equilibrium, the column 3 confluent (also known as the third protein product) is percolated five times its volume with 20 mM sodium phosphate, 50 mM sodium chloride, 1.5% (w / v) glycerol, pH 7.4. If the column 3 confluent concentration is < 8 g / L, the confluent is further concentrated to 8 g / L. After percolation (and concentration, if necessary), the confluent is recycled from the system using twice its system retrieval volume with 20 mM sodium phosphate, 50 mM sodium chloride, 1.5% (w / v) glycerol, pH 7.4. After recovery, a two-step dilution can be performed using the percolate solution. The target concentration for the first dilution is 6 g / L, and the target concentration for the second step is 5 g / L. Both steps can be used to achieve the target. If the concentration after the first dilution is within the target range, the second step may not be necessary.

[0207] Example 7: Intermediate Filtration and UF / DF 3

[0208] Cobalt-containing arginase 1 can be stored for extended periods, including long-term freezing, prior to the polyethylene glycolation reaction. The intermediate Co-arginase can be filtered through a 0.2 μm filter and can be frozen for long-term storage.

[0209] The UF / DF3 step uses a membrane with a molecular weight cutoff of 30 kDa. One function of this step is to perform buffer exchange and concentration on the filtered UF / DF2 confluence (fresh or thawed) to provide conditions best suited for polyethylene glycolation. If frozen Co-arginase 1 intermediate is used as the starting material, it will be thawed at room temperature for up to 36 hours. The membrane is sterilized with a cleaning solution (0.5 N NaOH) and rinsed with water. A standardized permeability test (NWP) is performed, and then the membrane is equilibrated before use in production. Once the UF / DF system reaches equilibrium, the Co-arginase 1 intermediate is percolated five times the percolation volume with 0.1 M sodium phosphate at pH 8.4. After percolation, the confluent is concentrated and recycled from the system using twice the system retrieval volume of 0.1 M sodium phosphate at pH 8.4. After recovery, a two-step dilution is performed using the percolation solution. The target concentration for the first dilution is 11 g / L, and the target concentration for the second dilution is 10 g / L. Two steps are used to facilitate reaching the target level. If the concentration after the first dilution is within the target range, the second step may not be necessary.

[0210] Regarding UF / DF 2 and UF / DF 3 steps, the first buffer exchange may be to 20 mM sodium phosphate, 50 mM NaCl, 1.5% glycerol, pH 7.4, ≥ 5 DV, and the protein is concentrated to approximately 5.0 mg / mL. The second buffer...The exchange can be performed in 0.1 M sodium phosphate, pH 8.1–8.5, and the protein is concentrated to approximately 10.0 mg / mL (in preparation for the PEGylation of drug A, page 23 / 39 of the drug information sheet, CN 121518448 A).

[0211] Example 8: PEGylation of arginase 1

[0212] PEGylation covalently links PEG (polyethylene glycol) to the Co-arginase 1 (active drug) molecule (see Table 4 for representative examples of the PEGylation step). In one embodiment, the PEGylation reaction covalently binds 5000 Da PEG molecules to Co-arginase 1. In alternative embodiments, PEGylation can be performed before cobalt substitution of arginase 1 or at other points in the manufacturing process. In one embodiment, the PEG conjugation reaction can use solid or liquid methoxyPEG succinimidyl carboxymethyl ester, which reacts with lysine in the space on Co-arginase 1. The resulting polyethylene glycol-modified protein (Co-rhARG1-PEG) has a molecular weight of approximately 280 kDa. The polyethylene glycol-modified conjugate can be filtered and stored at 2–8°C until the UF / DF4 operation.

[0213] Table 4: Polyethylene glycolation process of Co-rhARG1 active pharmaceutical ingredient

[0214]

[0215] In one embodiment, solid methoxy PEG succinimidyl carboxymethyl ester (MW 5000) can be added to a solution containing arginase 1 in 19.3 x mol excess and incubated for 0.5–4.0 hours at pH 8.4.

[0216] After polyethylene glycolation, unbound PEG is removed by ultrafiltration / percolation, arginase 1 is exchanged into a formulation buffer and concentrated for the formulation step. This UF / DF4 step uses a membrane with a molecular weight cutoff of 100 kDa. One function of this step is to exchange the PEG conjugate buffer into the final formulation while removing free PEG. The membrane used for this purpose is sterilized with a cleaning solution (0.5 N NaOH) and rinsed with water. Once the UF / DF system reaches equilibrium, the PEG conjugate is percolated against 5 mM potassium phosphate, 50 mM sodium chloride, 1.5% (w / v) glycerol, pH 7.4 for ten times the dialysis volume. After percolation, the conjugate is recovered from the system under pressure. Prior to the final filtration and filling steps, the recovered UF / DF4 conjugate is diluted to 5 g / L with 5 mM potassium phosphate, 50 mM sodium chloride, 1.5% (w / v) glycerol, pH 7.4. In an alternative embodiment (pages 24 / 39 of this specification, CN 121518448 A), arginase 1 is exchanged against 20 mM sodium phosphate, 50 mM sodium chloride, 1.5% (w / v) glycerol, pH 7.4.

[0217] In some embodiments, the formulation buffer of 5 mM potassium phosphate, 50 mM sodium chloride, 1.5% glycerol, pH 7.4, was adjusted to a protein concentration of approximately 5.0 mg / mL.

[0218] In some embodiments, it was found that the formulation buffer of 5 mM potassium phosphate, 50 mM sodium chloride, 1.5% glycerol, pH 7.4 enhanced the stability of arginase 1 during storage compared with other buffers such as sodium phosphate buffer. In one or more embodiments, the 5 mM potassium phosphate buffer contained 1 mM K2HPO4 and 4 mM KH2PO4.

[0219] The active pharmaceutical ingredient (Co-rhARG1-PEG) is a polyethylene glycol-modified cobalt-substituted human arginase 1 prepared by conjugating an activated PEG molecule with the ε-amino group of lysine and the amino group of the N-terminal amino acid. Dye-based fluorescence assays were used to determine the molar ratio of PEG molecules for each protein using phthalaldehyde. Phthalate reacts specifically with primary amines in the presence of thiols to form fluorescent derivatives. The measurement of fluorescence signals allows for the quantification of active free amines present in protein molecules. Quantification is based on a standard curve using N-acetylsine. The amount of polyethylene glycol-modified amines in each protein can be determined by subtracting the amount of free amines measured by fluorescence assay of the polyethylene glycol-modified drug from the theoretical amount of free amines present in unconjugated Co-arginase 1. The theoretical amount of free amines from lysine residues plus the N-terminal amino acid is 25. Free unconjugated PEG in the drug substance was measured by SEC-HPLC and by refractive index detection. The results can be expressed as µg / mL of free PEG (see Table 5).

[0219] Table 5: SEC-HPLC method parameters / free PEG / Co-rhARG1-PEG drug substance

[0220]

[0221] Example 9: CIEX-HPLC characterization of pharmaceutical intermediates

[0222] Various charge variants of arginase 1 may be generated during E. coli fermentation. Charge variants can be analyzed using a TSK gel cation exchange column via cation exchange HPLC (CIEX-HPLC). This analysis used mobile phase (A) 20 mM MES, pH 6.0 and mobile phase B 20 mM MES, 500 mM NaCl, pH 6.0; flow rate 1.0 mL / min; run time 40.0 min; column temperature 22°C; and mobile phase gradients according to Table 6.

[0223] Table 6: CIEX-HPLC Charge Variant Co-Arginine 1 Intermediate Gradient Program Specification 25 / 39 pages 31 CN 121518448 A

[0224]

[0225] Samples were diluted with formulation buffer prior to analysis. Results are described as percentage charge variant distribution. Figure 5(a) shows a representative chromatogram in which six major peaks of the Co arginase 1 intermediate are typically observed.

[0226] Example 10: iCIEF characterization of drug substance

[0227] The drug intermediate was polyethylene glycolated to form the drug substance. The polyethylene glycolation of the drug intermediate makes the use of the CIEX-HPLC method for the drug intermediate less suitable than other embodiments developed as part of this invention. Anion IEX-HPLC was evaluated, but adequate separation was not achieved. Alternatively, an imaging capillary isoelectric focusing (iCIEF) method was developed to analyze the charge variants of the drug substance.

[0228] In the presence of an applied electric field, the analyte in imaging capillary isoelectric focusing (iCIEF) migrates through the capillary via the reverse migration of hydrated hydrogen ions (anolyte) and hydroxide ions (cathode electrolyte). The sample was diluted in a matrix containing a carrier amphoteric electrolyte and a pI marker. Protein separation occurs in two focusing steps. An initial pre-focusing step establishes a pH gradient. In a second, higher-voltage focusing step, charge variants are more clearly focused and separated. UV absorption images of the entire capillary are digitally captured every 30 seconds and after the focusing step is completed.

[0229] The results can be expressed as a percentage distribution of charge variants. Figure 5(b) shows a representative electrophoresis plot where nine major peaks of the drug substance are observed. Peaks 3 and 4 are consolidated together because the resolution between these peaks has been shown to be variable. The relative areas of these peaks are provided in Table 7:

[0230] Table 7: iCIEF characterization of Co-rhARG1-PEG charge variants

[0231] Specification 26 / 39 pages 32 CN 121518448 A

[0232] Example 11: Enzyme activity of Co-arginase 1 intermediate and active pharmaceutical ingredient

[0233] An enzyme assay for measuring activity and identifying Co-arginase 1 intermediate and Co-rhARG1-PEG active pharmaceutical ingredient monitors the conversion of arginine to ornithine. The reaction mixture has one enzyme concentration tested at seven different arginine substrate concentrations in the range of 0 to 2 mM. The reaction is performed at 37°C for a fixed time. The reaction time has been established to ensure that substrate consumption is less than 10% at any given substrate concentration. The reaction is quenched, the product ornithine is derivatized, and quantified by reversed-phase UPLC.

[0234] Figures 8(a) (Co-arginase 1 intermediate) and 8(b) (Co-rhARG1-PEG active pharmaceutical ingredient) show examples of reaction rate versus substrate concentration plots and representative Kcat, Km, and Kcat / Km.

[0235] Examples12: Analysis of Cobalt and Manganese

[0236] Cobalt, residual manganese, and free cobalt were measured using inductively coupled plasma mass spectrometry (ICP-MS). Samples were digested by microwave, and all metals were released from the matrix using 1% nitric acid and 6% hydrogen peroxide. The resulting digests were analyzed by ICP-MS. Cobalt and residual manganese samples were digested without any sample treatment. Free cobalt was measured on permeate samples that had been ultrafiltered to separate the enzyme from the permeate, to measure cobalt independent of the enzyme. Table 8 summarizes some characteristics of the Co-arginase 1 intermediate.

[0237] Table 8: Typical characteristics of Co-arginase 1 intermediates

[0238]

[0239] Table 9: Typical characteristics of Co-rhARG1-PEG API 27 / 39 pages 33 CN 121518448 A

[0240]

[0241] Example 13: Post-translational modifications of Co-arginase 1 intermediates

[0242] Post-translational modifications of Co-arginase 1 intermediates were detected using a variety of techniques, such as peptide mapping analysis, LC-MS intact mass spectrometry, and reversed-phase LC / MS. Table 10 lists a summary of all identified modifications.

[0243] Table 10: Identified modifications of Co-arginase 1 intermediates 28 / 39 pages 34 CN 121518448 A

[0244]

[0245] Characterization determined that when Co-arginase 1 intermediate modifications were present, the primary modification was N-terminal gluconic acidification (confirmed by peptide mapping analysis). Additional characterization of arginase 1-modified substances was performed by testing samples collected at three time points: fermentation, post-column 1, and drug intermediates from column 3. The analytical methods typically require dissociation of arginase into monomers and analysis as monomers. N-terminal glucosylation of arginase 1 (analyzed as a monomer) was typically 10.8% to 13.9%. Other modifications were found in samples from the three time points as N-terminal phosphoglucosylated monomers (4.3% to 6.5%) and diglucosylated monomers (0.7% to 1.2%). In samples from a standardized reference production run, the levels of unmodified Co-arginase 1 (monomer) and Co-arginase 1 intermediates were comparable, ranging from 80.6% to 83.6%. Standard conditions used for the purification process (i.e., no salt gradient applied to column 1) moderately altered the relative levels of unmodified monomers carried into the Co-arginase 1 intermediate (81.1% to 83.6%).

[0246] Table 11: LC / MS results of Co-arginase 1 characterization

[0247]

[0248] Example 14: Changes in column 1 conditions

[0249] In an alternative embodiment, a NaCl gradient can be applied to column 1. Using a NaCl gradient on column 1 allows for the separation of different arginase 1 variants to select a preferred embodiment. Figure 7 shows a 0.0 to 0.2 M NaCl gradient applied to column 1. The fractions collected from the column 1 eluent were analyzed by SE-HPLC, CEX-HPLC, and RP-HPLC.

[0250] Using an analytical CEX-HPLC method, the peak numbers of the arginase 1 charge variants were designated as 1 to 6 (see page 29 / 39 of the specification, 35 CN 121518448 A, Figure 5(c)). The peak numbers are matched with various glucose acidification states and unglucose-acidified arginase 1. This analysis shows six peaks of arginase 1 eluted from the NaCl gradient. The arginase 1 variants designated as peak numbers 1, 2, and 3 eluted earlier in the column 1 eluent peaks. Peak 4 eluted at the highest concentration of arginase 1 eluted, and peaks 5 (unmodified arginase 1) and 6 eluted later in the elution peaks. Thus, different charge variants of arginase 1 were successfully separated using 0.0 to 0.2 M NaCl.

[0251] Alternative NaCl gradients, such as 0 to 0.5 M NaCl, can be used for column 1 elution. It was found that using a NaCl gradient reproducibly separated arginase 1 into six distinct peaks, thereby enabling the selection of specific arginase 1 variants for further processing in the manufacture of the drug substance or finished product.

[0252] Further analysis of the first protein product (and arginase 1 variants) was also performed by LC / MS (see Figure 6). LC / MS analysis identified the specific type of glucosylation generated by the production of arginase 1 in *E. coli*. LC / MS analysis identified unmodified arginase 1, glucosylated arginase 1, phosphoglucosylated arginase 1, and 2X glucosylated arginase 1.

[0253] Table 12 shows fractions with different glucosylation levels produced by applying a 0 to 0.2 M NaCl gradient (and corresponding fractions of CEX peaks 1 to 6). Each of peaks 1 to 6 was analyzed by LC / MS. The data showed that the main peak (peak 5) had a high percentage of unglucosylated arginase 1 and a high specific activity. Different fractions (corresponding to peaks 1 to 6) can be collected for further processing depending on the desired characteristics.

[0254] Table 12: LC / MS analysis of drug intermediate peaks 1 to 6.

[0255]

[0256] In addition to changing the NaCl on column 1In addition to concentration, different amounts of protein can be loaded onto column 1 to enhance the purification of non-glucosylated arginase 1 material.

[0257] Changing the loading factor of column 1 and using a NaCl gradient on column 1 can compensate for unexpected disturbances experienced during E. coli fermentation to produce glucosylated arginase 1 material.

[0258] Example 15: Variation of Fermentation Conditions

[0259] Experiments were conducted to determine the robustness of the fermentation conditions used to produce arginase 1. Table 13 shows that E. coli fermentation at a suboptimal pH of 7.6 produced more glucosylation than fermentation at the preferred pH of 7.2. Containers B1, B8, and B12 were used with optimal fermentation conditions: pH 7.2, dissolved oxygen 30%, and medium feed rate 0.06 mL / min. Container B3 was used to ferment E. coli expressing arginase 1 at pH 7.6 (above the optimal pH). The increase in pH resulted in a higher proportion of phosphogluconate adducts (23%, compared to 10-12% in the control run)

[0260] Table 13: Gluconate Arginase 1 Observed in Fermentation Vessels Instruction Manual 30 / 39 Pages 36 CN 121518448 A

[0261]

[0262] Example 16: Variation of Loading Factor in Column 1

[0263] Different amounts of E. coli cell lysate were applied to column 1 to determine the effect on the purification of arginase 1 charge variants and on yield and purity. Loading factors of 15 to 60 g protein / L resin were used under various conditions, and the variation of CIEX charge class curves is shown (Table 14). Higher loading factors resulted in better separation of gluconate variants (but depending on the fractions collected, this may result in a yield trade-off). For example, with a loading factor of 20 mg protein / mL resin, peak 5 was 45.8%, while it increased to 50.0% with a loading factor of 40 mg / mL.

[0264] Table 14: Effect of Column 1 Loading Factor on Protein Product 1 Specification 31 / 39 pages 37 CN 121518448 A

[0265]

[0266] Example 17: Phase 1 / 2 Clinical Study

[0267] The finished drug produced by the method of the present invention was used in a Phase 1 / 2 open-label study to evaluate the use of Co-rhARG1-PEG in arginase 1 deficiency and hyperarginemia. The primary endpoint of this study was to evaluate the safety and tolerability of intravenous (IV) administration of Co-rhARG1-PEG in subjects with hyperarginemia / arginase 1 deficiency. The secondary endpoint was: determining IVThe effect of the administered study drug on plasma arginine concentration; the effect of IV-administered study drug on plasma guanidine compounds (GC); and the pharmacokinetic (PK) curves of IV-administered study drug were characterized. Other endpoints included assessments of clinical outcomes in obtaining clinical benefit, such as: the 6-minute walk test (6MWT), gross motor function measurement (GMFM) parts D and E, and the adaptive behavior assessment system (ABAS).

[0268] Phase 1 / 2 data showed that Co-rhARG1-PEG was highly effective in sustainably reducing plasma arginine. Furthermore, control of patients' plasma arginine levels was accompanied by clinically significant responses in activity and adaptive behavior. Treatment is generally well tolerated. Hypersensitivity reactions are rare and can be controlled with standard measures.

[0269] The Co-rhARG1-PEG finished drug provided for the study was a liquid formulation packaged in a 10 mL disposable glass vial, containing 5 mL of the finished drug at a concentration of 1 mg / mL. The drug was formulated in 50 mM NaCl, 1 mM K2HPO4, 4 mM KH2PO4 and 1.5% w / v glycerol.

[0270] The Phase 1 / 2 study was conducted in two parts: Part 1 (single ascending dose) and Part 2 (repeated dosing). The study design of this Phase 1 / 2 trial 101A and 102A open-label extension is shown in the figure below:

[0271]

[0272] Part 1 involved patients trying the drug and focused on safety. Part 2 was designed to allow patients to receive a consistent dose and to look for biomarkers of clinical efficacy. Baseline assessment of arginine levels was performed before each part. All patients participating in Part 1 who meet the criteria for continued dosing may continue arginase 1 dosing in Part 2.

[0273] In this study, the starting dose received by each patient may be escalated in Part 1, with a 2-week clearance / observation period between each consecutive dose level. Possible doses for each patient in Part 1 are 0.015, 0.03, 0.06, 0.10, 0.15, 0.20, and 0.30 mg / kg, spaced 2 weeks apart as needed to optimize plasma arginine. Any specific dose may be repeated, or the dose may be increased / decreased between specified dose levels, if new data from previous dose levels meet certain criteria. For example, dose escalation may be terminated if one or more of the following criteria for dose escalation cessation are met: the patient's plasma arginine level is <40 for all samples collected over a period of at least 40 (± 2) hours following dosing.μM, or for all samples collected over a period of at least 112 (± 2) hours after administration, the patient’s plasma arginine level was <115 μM.

[0274] If none of these events occurred, the patient’s arginase 1 dose may be increased to the next higher dose every 2 weeks until any dose escalation cessation criteria are met or the patient has received the highest dose of 0.30 mg / kg under this regimen. Ultimately, the dose may also be increased beyond 0.30 mg / kg for therapeutic purposes.

[0275] Part 2 is the period of repeated dosing for patients who have completed Part 1. Part 2 found a dose and regimen for each patient that could safely optimize plasma arginine in the range of about 40 μM to about 115 μM during repeated dose administration, with an emphasis on maintaining pre-dose levels below 150–200 μM. Multiple dose levels may be used in Part 2 if data suggest that it is possible to better study dose-response outcomes during repeated dose administration. Arginine levels during treatment will also be compared to arginine levels determined before treatment.

[0276] Patients who completed Part 2 of 101A were eligible to participate in the Long-Term Open-Label Extension (OLE) Trial (NCT03378531). Treatment was administered with 24 weekly IV doses, with the option to switch to subcutaneous administration for the remainder of the 3-year OLE period.

[0277] Results Description 33 / 39 pages 39 CN 121518448 A

[0278] In all patients, the increase in mean Cmax and mean AUC0-168 was dose-proportional. For Co-rhARG1-PEG dose levels of 0.015, 0.03, 0.06, 0.1, and 0.2 mg / kg, the mean (± SD) Cmax were 0.428 ± 0.0915, 0.723 ± 0.247, 1.73 ± 0.538, 2.27 ± 0.238, and 6.13 (N=1) µg / mL, respectively. A slight anti-drug antibody (ADA) effect on mean Cmax was observed in both ADA-positive and ADA-negative patients (Figure 9).

[0279] Changes in AUC (AUC0-168, AUC0-∞) were dose-dependent within the studied dose range; note that no significant change was observed between 0.06 and 0.1 mg / kg (using available data). Mean clearance (CL) estimates ranged from 0.789 to 1.57 mL / hr / kg in all patients and from 0.776 to 1.33 mL / hr / kg in ADA-negative patients. Mean volume of distribution (Vss)The estimated values ​​ranged from 35.3 to 52.1 mL / kg in all patients and from 32.8 to 52.1 mL / kg in ADA-negative patients.

[0280] Part 1 of this study helped select the optimal (individual) starting dose for each patient in Part 2 using the observed PD (arginine) response. During week 1 of Part 2, mean circulating drug concentrations tended to increase in all patients using escalating doses of Co-rhARG1-PEG within the assessed dose range. After the first administration of Co-rhARG1-PEG in Part 2, mean Cmax increased proportionally to the dose in all patients. For Co-rhARG1-PEG dose levels of 0.015, 0.03, 0.04, 0.06, 0.09, 0.1, and 0.12 mg / kg, the mean (± SD) Cmax were 0.292 (N=1), 0.395 (N=1), 1.01 ± 0.221, 1.75 ± 0.391, 1.99 (N=1), 2.34 (N=1), and 2.87 ± 0.626 µg / mL, respectively.

[0281] In week 8 of Part 2, the mean circulating drug concentration generally increased with increasing doses of Co-rhARG1-PEG in all patients. At week 8, there was no significant ADA effect on available PK concentrations. As a result of this data, it was assumed that most (13 / 14) patients had reached a stable state at this point. Following the 8th QW dose of Co-rhARG1-PEG, the increase in mean Cmax and AUC0-168 was dose-proportional in all patients.

[0282] In addition to pharmacokinetic data, pharmacodynamic (arginine) data were also collected (Figure 10). In Part 2, patients with arginase 1 deficiency were administered (weekly) QW IV doses of Co-rhARG1-PEG, and in Part 1, the starting dose was selected based on the observed PD (arginine) response. Following the first QW IV dose of Co-rhARG1-PEG, circulating arginine levels decreased significantly, particularly for Co-rhARG1-PEG doses equal to or greater than 0.04 mg / kg. In some cases, individual arginine concentrations decreased to below 40 µM. Furthermore, most patients did not fully recover to their initial arginine levels at doses ≥ 0.04 mg / kg and immediately before the administration of the second QW (weekly) dose of Co-rhARG1-PEG.

[0283] Overall, exposure to Co-rhARG1-PEG generally increases, and arginine inhibition increases with increasing dose. In the firstIndividualized dosing optimization was performed in the second part, resulting in a different number of patients at each dose level in weeks 1 and 8 of the second part.

[0284] Example 18: Subcutaneous Administration

[0285] After the completion of the second part of the phase 1 / 2 study in Example 17, some patients were switched from IV administration of Co-rhARG1-PEG to subcutaneous administration. Surprisingly, subcutaneous administration of Co-rhARG1-PEG gave a pharmacodynamic profile that appeared to be superior to that of IV administration. Also unexpectedly, the same formulation used for IV administration was successfully used for subcutaneous administration of Co-rhARG1-PEG.

[0286] Subcutaneous administration of Co-rhARG1-PEG maintained patients’ arginine levels within the preferred (healthy) target range of plasma arginine concentration for a longer duration than IV administration (Figure 11). The preferred optimized plasma arginine concentration for patients is in the range of about 40 μM to about 115 μM (during repeated dose administration), with the focus on maintaining levels below 150–200 μM pre-administration. As shown in Figure 11, subcutaneous administration of Co-rhARG1-PEG resulted in arginine concentrations above the lower level of 40 μM (page 34 / 39 of the specification, 40 CN 121518448 A) and below the higher level of 115 μM. Surprisingly, subcutaneous administration yielded arginine concentrations entirely within the preferred range. This means that patients will remain within the appropriate plasma arginine concentration range until receiving another weekly dose of Co-rhARG1-PEG.

[0287] Example 19: Pharmacodynamics and Clinical Response of Phase 1 / 2 Clinical Study and Open-Label Extension

[0288] Sixteen patients (11 children and 5 adults) were enrolled in Part 1 of 101A, and 15 patients were enrolled in Part 2 of 101A. Two patients withdrew from the trial for personal reasons (one withdrew after dose 3 of Part 1, and one withdrew after dose 3 of Part 2). All 14 patients who completed Part 2 of 101A were enrolled in the OLE trial.

[0289] Patient baseline characteristics are shown in Table 15.

[0290] Table 15: Baseline Characteristics

[0291]

[0292] Analysis of plasma arginine and guanidine compound levels revealed a significant and sustained decrease in plasma arginine levels (Figure 12(a) shows a median decrease of 274 µM relative to baseline after 20 doses of polyethylene glycol arginase. The decrease in plasma arginine from baseline to dose 1, dose 8, and OLE was statistically significant (p<0.001). The decrease in plasma arginine was accompanied by a decrease in plasma guanidine compound (GC) levels. Figure 12(b)Plasma levels of guanidinoacetic acid (GAA), N-α-acetyl-L-arginine (NAA), α-keto-δ-guanidinopentanoic acid (GVA), and arginine (ARGA) at baseline, and a decrease in plasma GC levels during OLE, are shown.

[0293] 15 of the 16 patients completed all activity assessments at baseline (Patient 13 was wheelchair-bound) (Figure 13(a)). Deficits were defined as: 6MWT: less than the 5th percentile below; GMFMD: < 35 out of 39; GMFME: < 68 out of 72; ABAS-3: ≤ 85. 88% of the patients (14 of 16) had at least one activity deficit at baseline; 88%, 50%, and 56% of the 16 patients were classified as having baseline deficits in the 6MWT, GMFMD, and GMFME, respectively. Ten patients were eligible for the ABAS-3 assessment of adaptive behavior at baseline. Six patients were not tested for technical reasons (including limitations due to language, age, and cognitive impairment), and eight of the ten patients (80%) had baseline deficits in one or more domains assessed by ABAS-3.

[0294] Overall clinical response showed that, based on an improvement of ≥1 MCID in at least one of the 6MWT, GMFM-D, or GMFM-E assessments, 11 of the fourteen patients (79%) were defined as responders at dose 20 (Figure 13(b)). The 20-dose data indicated that 6MWT, GMFM-D, and GMFM-E were sufficiently sensitive to changes in clinical benefit in ARG1-D patients. The percentage of overall responders increased significantly from dose 8 to dose 20. All five patients (100%) who reached dose 44 maintained their overall clinical response status as responders at dose 20. Instructions for Use, pages 35 / 39, 41 CN 121518448 A

[0295] All respondents to a single assessment scale had an improvement of ≥1 MCID (Figures 13(b) and 14). For 6MWT, 7 out of 13 patients (54%) responded to this assessment scale only. The mean change in 6MWT was 32 meters in all patients and 66 meters in the 7 respondents. For GMFM-D: 5 out of 8 patients with baseline defects (63%) responded to this assessment scale only (mean MCID 1.84, range 1.21 to 3.33). For GMFM-E: 5 out of 8 patients with baseline defects (63%) responded to this assessment scale only (mean MCID 1.84, range 1.21 to 3.33).The MCID was 4.79 (range 1.67 to 8.33). At dose 20, the percentage of responders on individual activity assessment scales was significantly higher than at dose 8.

[0296] Data from all patients after 20 doses of PEGylated arginase showed a significant and sustained decrease in plasma arginine, improvement in important disease outcomes, and a clinical responder rate of 79%. Phase 1 / 2 and OLE trials demonstrated the value of using 6MWT, GMFM-D, or GMFM-E as tools to obtain the clinical benefit of PEGylated arginase. PEGylated arginase is well tolerated, and the incidence of treatment-related adverse events decreased over time. Evidence of improved arginine control and clinical benefit following PEGylated arginase treatment further validated the key endpoints and design elements of the pivotal Phase 3 PEACE trial (NCT03921541).

[0297] Example 20: Phase 3 Clinical Trial Design

[0298] A randomized, double-blind, placebo-controlled Phase 3 study is being conducted using Co-rhARG1-PEG produced by the method of the present invention to investigate the efficacy and safety of Co-rhARG1-PEG in children and adults with arginase 1 deficiency. The trial is currently ongoing as a PEACE (CAEB1102-300A; NCT03921541).

[0299] The study design for this Phase 3 trial is shown in the figure below:

[0300]

[0301] Key inclusion criteria

[0302] a. Age ≥ 2 years, diagnosed with ARG1-D and plasma arginine level ≥ 250 μmol / L, to statistically test the proportion of patients achieving plasma arginine levels below the medical guidance value of 200 μmol / L

[0303] b. Able to maintain a stable and consistent diet during the blind trial

[0304] c. Able to maintain a stable dose of ammonia scavengers, antiepileptic therapy and / or medications for seizures during the blind trial

[0305] d. Able to perform and successfully complete clinical assessments and must have baseline deficiencies in one of the assessment scales for secondary clinical response endpoints, as shown in Table 16.

[0306] Key Exclusion Criteria

[0307] a. An episode of hyperammonemia requiring hospitalization within 6 weeks prior to the start of treatment

[0308] b. An active infection within 3 weeks prior to receiving the first dose of PEGylated arginase

[0309] c. Extreme incapacity, defined as inability to perform an assessment on the Gillette Functional Assessment Questionnaire (GFAQ) or a GFAQ score of 1 (completely unable to complete any steps). (Instructions for Use 36 / 39)Page 42 CN 121518448 A

[0310] d. Involved in previous polyethylene glycol arginase intervention studies or currently involved in other clinical trials

[0311] e. History of polyethylene glycol allergy

[0312] Table 16: Definition of baseline defects for key clinical response endpoints

[0313]

[0314] 2MWD = 2-minute walk distance; GMFM = gross motor function measurement; Part D = standing; Part E = walking, running, jumping

[0315] *NIH Toolbox (U.S. Department of Health and Human Services, Washington, D.C.) Motor Domain Dataset (2-minute walk endurance test)

[0316] The primary endpoint of this Phase 3 trial was a reduction in plasma arginine (based on changes from baseline in treatments, changes in plasma arginine levels at week 24 in individual patients in the active and placebo groups).

[0317] Secondary endpoint measures included:

[0318] a. Clinical response endpoint: Clinically responsive patients were defined as those who showed improvement in at least one of the 2MWT, GMFM-D, or GMFM-E clinical response endpoints at week 24, as defined in Table 17

[0319] b. Response rate for each individual assessment scale of the clinical response endpoint

[0320] c. Other clinical outcome assessments

[0321] i. Functional Activity Scales 5, 50, and 500

[0322] ii. Gillette Functional Assessment Questionnaire (GFAQ)

[0323] iii. Vinland Adaptive Behavior Scale – II

[0324] d. Safety assessment, including immunogenicity

[0325] e. Proportion of patients with plasma arginine < 200 μM and within the normal range (40–115 μM)

[0326] f. Pharmacokinetic characteristics of PEGylated arginase

[0327] Table 17: Definition of Clinical Response Endpoints for Clinically Responding Participants

[0328]

[0329] The total duration of the study is expected to be approximately 178 weeks per participant, including a long-term open-label extension period (3 to 4 weeks of screening, 24 weeks of treatment, followed by a long-term open-label extension period of up to 150 weeks). Participants will receive weekly IV infusions (approximately 30 minutes) of Co-rhARG1-PEG or a volume-adjusted placebo once a week. Dosage modifications of Co-rhARG1-PEG based solely on plasma arginine levels will be performed by an informed pharmacist and / or physician according to a dosing algorithm. After the first 8 weeks of the blinded long-term extension period, participants may opt to receive Co-rhARG1-PEG via subcutaneous administration, subject to approval from the investigator and sponsor. The initial mg / kg subcutaneous dose may be the same as the IV dose.

[0330] Allocation of Co-rhARG1-PEGSubjects started at dose level 2 (see Table 18 below), i.e., 0.10 mg / kg. Starting from the 5th visit, if necessary, the informed physician would modify the dose based on plasma arginine levels according to the following dosing algorithm:

[0331] • If the plasma arginine level is >150 μM, the dose will be increased by 2 dose levels (not exceeding 0.20 mg / kg) using a single 168-hour sample, provided that the two doses prior to this sample were a) the same dose level in mg / kg and b) consecutive (without missed doses).

[0332] • If the plasma arginine levels from two consecutive 168-hour samples are both <50 μM (regardless of whether there are missed doses), the dose will be reduced by 1 dose level (see Table 17), but not to below 0.05 mg / kg.

[0333] Table 18: Dosage Adjustment of Co-rhARG1-PEG

[0334]

[0335] Statistical Considerations

[0336] The preliminary analysis will be based on the mean of the last four plasma arginine measurements that met strict pre-specified criteria, after 24 weekly doses, comparing the mean decrease in plasma arginine levels from baseline in patients treated with PEG arginase with that in patients treated with placebo.

[0337] Using a two-sided Mann-Whitney-Wilcoxon test, assuming a common SD of 120 μM, sample sizes of 10 and 20 patients, respectively, randomly assigned to placebo and PEG arginase, respectively, achieved 98% efficacy to demonstrate a mean difference in plasma arginine levels of 200 μM at a significance level of 0.05.

[0338] Furthermore, this number of subjects provided more than 80% efficacy, and the statistically significant difference in the proportion of groups at the clinical response endpoint was 40% at a significance level of 0.05, as detected by Fisher's exact test.

[0339] Example 21: Site-Specific Polyethylene Glycolization Analysis

[0340] Figure 15 provides an exemplary site-specific polyethylene glycolization analysis. Three Co-rhARG1-PEG batches were analyzed. Peptide mapping analysis was performed as follows: Co-rhARG1-PEG active pharmaceutical ingredient, Co-Arginase 1 intermediate (non-polyethylene glycolized) batches, and corresponding reference standards were denatured with guanidine-HCl, reduced with DTT, and alkylated with iodoacetamide. These samples were diluted with 50 mM Tris buffer 8.0. Each sample was digested with sequencing-grade trypsin at 37°C for approximately 5 hours. Using an acetonitrile gradient in 0.05% trifluoroacetic acid,The peptides were resolved on a Waters Acquity ВЕН 300 C18 column, 2.1 × 150 mm, Waters C / N 186003687. LC-MS and MS / MS cleavage of the peptides were obtained on a Waters Xevo G2-XS QTOF MS / MS.

[0341] It can be seen that none of the three batches exhibited polyethylene glycolation at sites K3, K149, K190, K195, K29, K265, or K283. Furthermore, all three batches exhibited polyethylene glycolation at sites K16, K32, K38, K40, K47, K67, K74, K82, K87, K88, K152, K154, K171, K312, and K321. Some batches showed lower polyethylene glycolation frequencies at sites K222 and K223.

[0342] References throughout this specification to “one embodiment,” “some embodiments,” “various embodiments,” “one or more embodiments,” or “embodiment” are intended to refer to a particular feature, structure, material, or property described in connection with that embodiment, which is included in at least one embodiment of this disclosure. Therefore, words such as “in one or more embodiments,” “in some embodiments,” “in various embodiments,” “in one embodiment,” or “in an embodiment” appearing in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Furthermore, specific features, structures, materials, or properties may be combined in any suitable manner in one or more embodiments.

[0343] Although the disclosure herein provides for a description with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of this disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to this disclosure without departing from its spirit and scope. Therefore, this disclosure is intended to include modifications and variations within the scope of the appended claims and their equivalents. Instruction manual, pages 39-39, 45 CN 121518448 A, Figure 1(a), Figure 1(b), Figure 1(c); Instruction manual, Figure 1 / 22, page 46 CN 121518448 A, Figure 2; Instruction manual, Figure 2 / 22, page 47 CN 121518448 A, Figure 3(a); Instruction manual, Figure 3 / 22, page 48 CN 121518448 A, Figure 3(b); Instruction manual, Figure 4 / 22, page 49 CN 121518448 A, Figure 4(a); Instruction manual, Figure 5 / 22, page 50 CN 121518448 A, Figure 4(b); Instruction manual, Figure 6 / 22, page 51 CN 121518448 AFigure 4(c) Figure 5(a) Appendix 7 / 22 Page 52 CN 121518448 A Figure 5(b) Figure 5(c) Appendix 8 / 22 Page 53 CN 121518448 A Figure 5(d) Figure 6 Appendix 9 / 22 Page 54 CN 121518448 A Figure 7 Figure 8(a) Appendix 10 / 22 Page 55 CN 121518448 A Figure 8(b) Figure 9(a) Appendix 11 / 22 Page 56 CN 121518448 A Figure 9(b) Figure 9(c) Appendix 12 / 22 Page 57 CN 121518448 A Figure 9(d) Figure 9(e) Appendix 13 / 22 Page 58 CN 121518448 A Figure 9(f) Figure 10(a) Appendix 14 / 22 Page 59 CN 121518448 A Figure 10(b) Figure 10(c) Appendix to the Instruction Manual Page 15 / 22 60 CN 121518448 A Figure 11(a) Figure 11(b) Appendix to the Instruction Manual Page 16 / 22 61 CN 121518448 A Figure 12(a) Appendix to the Instruction Manual Page 17 / 22 62 CN 121518448 A Figure 12(b) Appendix to the Instruction Manual Page 18 / 22 63 CN 121518448 A Figure 13(a) Appendix to the Instruction Manual Page 19 / 22 64 CN 121518448 A Figure 13(b) Figure 14(a) Appendix to the Instruction Manual Page 20 / 22 65 CN 121518448 A Figure 14(b) Figure 14(c) Appendix to the Instruction Manual Page 21 / 22 66 CN 121518448 A Figure 15 Figure 22 / 22 Page 67 CN 121518448 A Abstract Described are methods for producing recombinant Arginase, such as PEGylated, cobalt-substituted recombinant human Arginase 1. Also described are pharmaceutical compositions comprising such recombinant Arginase, as well as methods of treatmentand uses of such recombinant Arginase.

Claims

1. A method for producing purified recombinant cobalt-substituted human arginase, wherein the recombinant human arginase (rhARG) comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1, the method comprising: a. Culturing E. coli cells that produce rhARG in a bioreactor; b. Lyse the E. coli cells; c. Remove cell debris from the lysate; d. Load the cell lysate onto a cation exchange column; e. Elute the rhARG with a high-salt solution; f. Incubate the eluted rhARG with cobalt salt to form cobalt-substituted rhARG (Co-rhARG); g. Apply the Co-rhARG to an anion exchange column and collect the flow-through; h. Apply the flow-through to the third chromatographic column; as well as i. Elute the Co-rhARG from the third chromatographic column with a high-salt solution.

2. The method of claim 1, wherein up to 60 grams of rhARG per liter of cation exchange resin is loaded onto the cation exchange column.

3. The method according to claim 1 or 2, wherein the rhARG is eluted from the cation exchange column using a high-salt solution with a salt concentration of up to about 0.5 M.

4. The method according to claim 1 or 2, wherein the rhARG is eluted from the cation exchange column using a high-salt solution with a salt concentration of about 0.1 M.

5. The method according to claim 1 or 2, wherein the rhARG is eluted from the cation exchange column using a salt concentration gradient of about 0.0 to about 0.5 M.

6. The method of claim 1 or 2, wherein the rhARG is eluted from the cation exchange column using a salt concentration gradient of about 0.0 to about 0.2 M.

7. The method according to any one of claims 1 to 6, wherein the cobalt salt comprises Co. 2+ Salt.

8. The method according to any one of claims 1 to 7, wherein the cobalt salt comprises CoCl2.

9. The method according to any one of claims 1 to 8, wherein the third chromatographic column comprises a multimode chromatography (MMC) column.

10. The method according to any one of claims 1 to 9, further comprising reacting the rhARG or Co-rhARG with a polyethylene glycolation reactant to provide a polyethylene glycolated protein.

11. The method of claim 10, wherein the polyethylene glycol-modified protein comprises one or more of the polyethylene glycol-modified amino acid residues at K16, K32, K38, K40, K47, K67, K74, K82, K87, K88, K152, K154, K171, K222, K223, K312, and K321.

12. The method of claim 11, wherein the PEGylated protein comprises one or more of the following being PEGylated: about 15% to about 60% K16, about 35% to about 80% K32, about 20% to about 85% K38, about 10% to about 60% K40, about 10% to about 60% K47, about 40% to about 90% K67, about 30% to about 95% K74, about 30% to about 98% K82, about 15% to about 65% K87, about 25% to about 70% K88, about 25% to about 85% K152, about 15% to about 65% K154, about 20% to about 75% K171, 0% to about 30% K222, 0%... From approximately 35% of K223, from approximately 0% of approximately 45% of K312, and from approximately 0% of approximately 45% of K321.

13. The method according to any one of claims 10 to 12, wherein the polyethylene glycol-modified protein contains polyethylene glycol-modified amino acid residues at least at K16, K32, K38, K40, K47, K67, K74, K82, K87, K88, K152, K154, K171, K312, and K321.

14. The method according to any one of claims 10 to 13, wherein the polyethylene glycol-modified protein does not have polyethylene glycol-modified amino acid residues at K3, K149, K190, K195, K29, K265 and K283.

15. A method for producing purified polyethylene glycol-modified recombinant cobalt-substituted human arginase, wherein the recombinant human arginase (rhARG) comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1, the method comprising: a. Cultivating Escherichia coli that produce rhARG in a bioreactor; b. Lyse the E. coli cells; c. Remove cell debris from the lysate; d. Load the cell lysate onto a cation exchange column; e. Elute the rhARG with a high-salt solution; f. Incubate the eluted rhARG with 10 mM CoCl2 to form cobalt-substituted rhARG (Co-rhARG); g. Apply the Co-rhARG to the anion exchange column and collect the flow-through; h. Apply the flow-through to a multimode chromatography (MMC) column; i. Elute the Co-rhARG from the MMC column with a high-salt solution; j. Add an excess of methoxyPEG succinimide carboxymethyl ester; k. Remove excess PEG.

16. A method for producing purified recombinant cobalt-substituted human arginase, wherein the recombinant human arginase (rhARG) comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1, the method comprising: a. In a bioreactor, Escherichia coli cells producing rhARG were cultured with agitation and aeration at a temperature between about 36°C and about 38°C and a pH between about 7.0 and about 7.

4. i. Adjust the temperature of the bioreactor to approximately 29°C; ii. Inducing E. coli cells to produce rhARG; iii. Culture the E. coli cells for approximately 18 hours; iv. Harvest the *E. coli* cells by centrifugation; b. The *E. coli* cells were lysed by high-pressure homogenization at a pH between approximately 7.2 and approximately 7.6 and at approximately 15°C or lower in 25 mM HEPES. c. Remove cell debris from the lysate by centrifugation at 15°C or lower; The pyrolysis product was then filtered using a 0.8-micron filter, followed by a 0.5-micron filter. d. Load the cell lysate onto a cation exchange column, and then wash the column with 25 mM HEPES at pH 7.2-7.6; e. Elute the rhARG at room temperature with a high-salt solution containing 25 mM HEPES and 0.1 M NaCl, pH 7.2–7.6; f. Incubate the eluted rhARG with 10 mM CoCl2 at room temperature for about 2 to about 8 hours to form cobalt-substituted rhARG (Co-rhARG); i. Exchange Co-rhARG into 50 mM Tris, pH 8.1-8.5; g. Apply the Co-rhARG to an anion exchange column and collect the flow-through; h. Apply the flow-through to a Capto multimode chromatography (MMC) column; i. Elute the Co-rhARG from the MMC column using a high-salt solution containing 50 mM Tris and 250 mM NaCl, pH 8.1-8.

5.

17. A method for producing purified recombinant polyethylene glycol-modified recombinant cobalt-substituted human arginase, wherein the recombinant human arginase (rhARG) comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1, the method comprising: a. In a bioreactor, E. coli cells producing rhARG were cultured with agitation and aeration at approximately 36°C and 38°C and at a pH between approximately 7.0 and approximately 7.

4. i. Adjust the temperature of the bioreactor to approximately 29°C; ii. Inducing Escherichia coli to produce rhARG; iii. Incubate the *E. coli* for approximately 18 hours; iv. Harvest the E. coli by centrifugation; b. The *E. coli* cells were lysed by high-pressure homogenization at a pH of about 7.2 to about 7.6 and at a temperature of about 15°C or lower in 25 mM HEPES; c. Remove cell debris from the lysate by centrifugation at 15°C or lower; The pyrolysis product was then filtered through a filter of approximately 0.8 micrometers, followed by a filter of approximately 0.5 micrometers. d. Load the cell lysate onto a cation exchange column, and then wash the column with 25 mM HEPES at pH 7.2-7.6; e. Elute the rhARG at room temperature with a high-salt solution containing 25 mM HEPES and 0.1 M NaCl, pH 7.2–7.6; f. Incubate the eluted rhARG with 10 mM CoCl2 at room temperature for about 2 to about 8 hours to form cobalt-substituted rhARG (Co-rhARG); i. Exchange Co-rhARG into 50 mM Tris, pH 8.1-8.5; g. Apply the Co-rhARG to an anion exchange column and collect the flow-through; h. Apply the flow-through to a Capto multimode chromatography (MMC) column; i. Elute the Co-rhARG from the MMC column with a high-salt solution containing 50 mM Tris and 250 mM NaCl, pH 8.1-8.5 (MMC buffer); i. Replace the MMC buffer with 20 mM sodium phosphate, 50 mM NaCl, 1.5% glycerol, pH 7.4 (buffer 1), and adjust the protein concentration to approximately 5.0 mg / mL; ii. Replace buffer 1 with 0.1 M sodium phosphate, pH 8.1-8.5 (buffer 2), and concentrate the protein concentration to approximately 10.0 mg / mL; j. Add an excess of methoxyPEG succinimide carboxymethyl ester with a molecular weight of approximately 5,000 Da, wherein approximately 19 moles of methoxyPEG succinimide carboxymethyl ester are added for every mole of protein, and incubate this mixture at approximately pH 8.4 for approximately 30 minutes to approximately 4 hours. k. Excess PEG was removed by exchanging buffer 2 with 20 mM sodium phosphate, 50 mM NaCl, 1.5% glycerol, pH 7.

4.

18. A composition comprising Co-rhARG or Co-rhARG-PEG produced by the method according to any one of claims 1 to 17.

19. The composition of claim 18, wherein the protein is covalently linked to polyethylene glycol at one or more of K16, K32, K38, K40, K47, K67, K74, K82, K87, K88, K152, K154, K171, K222, K223, K312, and K321.

20. A composition comprising a recombinant human arginase (rhARG) protein, wherein the protein comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1, wherein the protein is complexed with a non-natural metal cofactor, wherein the non-natural metal cofactor is cobalt, and wherein the protein is covalently linked to polyethylene glycol at one or more of K16, K32, K38, K40, K47, K67, K74, K82, K87, K88, K152, K154, K171, K222, K223, K312, and K321.

21. The composition according to any one of claims 18 to 20, wherein the recombinant human arginase (rhARG) comprises amino acid substitutions at positions selected from the group consisting of: H100, D123, H125, D127, D231, D233, W121, D180, S229, C302, and E255.

22. The composition according to any one of claims 18 to 21, wherein the recombinant human arginase (rhARG) comprises at least one amino acid substitution selected from the group consisting of: D180S, S229C, S229G, C302F, C302I, E255Q, D180E, and S229A.

23. The composition according to any one of claims 18 to 22, wherein the recombinant human arginase (rhARG) comprises at least one amino acid substituted with C302.

24. The composition according to any one of claims 18 to 23, wherein the recombinant human arginase (rhARG) comprises at least two amino acid substitutions.

25. The composition according to any one of claims 18 to 24, wherein the recombinant human arginase (rhARG) is a truncated arginase I protein.

26. The composition according to any one of claims 18 to 25, wherein the recombinant human arginase (rhARG) further comprises an exogenous protein fragment.

27. The composition of claim 26, wherein the exogenous protein fragment comprises the Fc region of an immunoglobulin or a portion thereof.

28. The composition according to any one of claims 18 to 27, wherein the specific activity of Co-rhARG-PEG is in the range of about 400 U / mg to about 700 U / mg.

29. The composition according to any one of claims 18 to 28, wherein, when measured in vitro, the protein exhibits a response to arginine hydrolysis at pH 7.4 at about 200 mM. -1 s -1 Approximately 4,000 mM -1 s -1 k within the range cat / K m .

30. The composition of claim 29, wherein, when measured in vitro, the protein exhibits a response to arginine hydrolysis at pH 7.4 at approximately 400 mM. -1 s -1 Approximately 2,500 mM -1 s -1 k within the range cat / K M .

31. The composition according to any one of claims 18 to 30, wherein the molar ratio of PEG:Co-rhARG is in the range of about 7 mol / mol to about 15 mol / mol.

32. The composition according to any one of claims 18 to 31, wherein the free PEG concentration is less than or equal to 100 µg / mL.

33. The composition according to any one of claims 18 to 32, wherein the total cobalt content in the composition is in the range of about 9 µg / mL to about 15 µg / mL.

34. The composition according to any one of claims 18 to 33, wherein when the composition is loaded onto an imaging capillary isoelectric focusing (iCIEF), at least nine peaks are generated, wherein peak 1 is less than 20%, peak 2 is less than 30%, peaks 3+4 are in the range of 10-30%, peak 5 is in the range of 15-30%, peak 6 is in the range of 10-25%, peak 7 is less than 25%, peak 8 is less than 15%, and peak 9 is less than 8%.

35. The composition according to any one of claims 18 to 34, wherein when the composition is loaded onto icIEF, at least nine peaks are generated, wherein peak 1 is in the range of 5-7%, peak 2 is in the range of 8-11%, peaks 3+4 are in the range of 16-20%, peak 5 is in the range of 21-24%, peak 6 is in the range of 21-22%, peak 7 is in the range of 14-15%, peak 8 is in the range of 5-8%, and peak 9 is in the range of 2-3%.

36. A pharmaceutical composition comprising Co-rhARG or Co-rhARG-PEG according to any one of claims 18 to 35 and a pharmaceutical carrier.

37. The pharmaceutical composition of claim 36, wherein the composition is formulated for intravenous or subcutaneous administration.

38. The pharmaceutical composition according to claim 36 or 37, wherein the composition comprises potassium phosphate, sodium chloride and glycerol.

39. The pharmaceutical composition according to any one of claims 36 to 38, wherein the composition comprises about 50 mM NaCl, about 1 mM K2HPO4, about 4 mM KH2PO4 and about 1.5% w / v glycerol.

40. A method for treating arginase 1 deficiency, the method comprising administering to a patient a pharmaceutical composition according to any one of claims 36 to 39.

41. The method of claim 40, wherein the pharmaceutical composition is administered intravenously.

42. The method of claim 40, wherein the pharmaceutical composition is administered subcutaneously.

43. The method according to any one of claims 40 to 42, wherein the pharmaceutical composition is initially administered at a dose of 0.1 mg / kg based on the weight of the unpolyglycolated enzyme.

44. The method according to any one of claims 40 to 43, further comprising monitoring the patient's plasma arginine level.

45. The method according to any one of claims 40 to 44, wherein the dose is adjusted according to the following algorithm: a. If the plasma arginine level is >150 μM, the dose will be increased by two dose levels in the table below (not exceeding 0.20 mg / kg) using a single 168-hour sample, provided that the two doses prior to this sample were a) the same dose level in mg / kg and b) consecutive (without missing doses). b. If the plasma arginine levels from two consecutive 168-hour samples (regardless of whether a dose was missed) are both <50 μM, then reduce the dose by one dose level in the table below, but not below 0.05 mg / kg; 。

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