Production method and application of human recombinant arginase 1

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

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 may also be used to treat cancer. Background Technology

[0002] Arginase 1 deficiency, or hyperarginemia, is a rare amino acid metabolic disorder caused by a lack 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 primarily 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 RBCs (< 1% of normal).

[0004] Current treatment for arginase 1 deficiency focuses on maintaining plasma arginine levels as close to normal as possible through lifelong dietary protein restriction. Protein intake is limited to the minimum necessary for protein biosynthesis and growth. Half or more of the dietary protein is provided as 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 limited treatment options for patients with arginase 1 deficiency highlight a significant unmet need for therapies that reduce arginine levels to the normal range and promote lifelong maintenance of normal arginine levels. The development of such treatments could help minimize the neurotoxic effects of arginine and its metabolites on patients 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 also be used to treat other diseases. 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] Arginase production

[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; shown in Figure 1(a)). In other embodiments, the recombinant human arginase protein is recombinant human arginase 2 (rhARG2) (SEQ ID NO: 3; 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.

[0009] Human arginase 1 and 2 proteins subjected to the methods of the present invention have two Mn 2+ Sites; one or two sites may be substituted to produce arginase 1 or 2 proteins modified with non-natural metal cofactors. In some embodiments, the protein exhibits a concentration greater than 200 mM at pH 7.4. -1 s -1 k cat / K M In a specific embodiment, the protein exhibited a concentration of approximately 200 mM at pH 7.4. -1 s -1 Approximately 4,000 mM -1 s -1 k within the range cat / K M In another embodiment, the protein exhibited a concentration of approximately 400 mM at pH 7.4 and 37°C. -1 s -1 Approximately 2,500 mM -1 s -1 k within the range cat / K M In certain embodiments, the present invention contemplates a protein comprising the amino acid sequence of human arginase 1 or 2 and a non-natural metal cofactor, wherein said protein exhibits a concentration greater than 400 mM at 37°C and pH 7.4. -1 s -1 k cat / K M An exemplary k at pH 7.4 and 37°C. cat / K M Values ​​include approximately 200, approximately 250, approximately 300, approximately 350, approximately 400, approximately 450, approximately 500, approximately 550, approximately 600, approximately 650, approximately 700, approximately 800, approximately 900, approximately 1,000, approximately 1,100, approximately 1,200, approximately 1,500, approximately 2,000, approximately 2,500, approximately 3,000, approximately 3,500, and approximately 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 steps of: 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 *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 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 eluting the recombinant human arginase protein (rhARG) from a cation exchange column using a high-salt solution with a salt concentration of up to about 0.5 M. In some embodiments, the recombinant human arginase protein (rhARG) is eluted from the cation exchange column using a high-salt solution with a concentration of 0.1 M. In some embodiments, the recombinant human arginase protein (rhARG) is eluted from the cation exchange column using a salt concentration gradient of about 0.0 to about 0.5 M. In some embodiments, the recombinant human arginase protein (rhARG) is eluted from the cation exchange column using a salt concentration gradient of about 0.0 to about 0.2 M.

[0014] In one or more embodiments, a method for producing recombinant cobalt-substituted human arginase protein (Co-rhARG) includes eluting recombinant human arginase protein (rhARG) from a cation exchange column with a mixture containing Co. 2+ The cobalt salt is incubated together with the cobalt salt. In some embodiments, the cobalt salt includes CoCl2.

[0015] In one or more embodiments, a method for producing recombinant cobalt-substituted human arginase protein (Co-rhARG) includes adding a recombinant cobalt-substituted human arginase protein (Co-rhARG) flowstream to a third chromatographic column comprising a multimode chromatography (MMC) column.

[0016] In one or more embodiments, a method of 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 glycolated protein contains one or more polyethylene glycolated 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, 0% to about 30% of K222, and 0% to about 35% of K222. The protein contains K223, 0% to about 45% of K312, and 0% to about 45% of K321. In some embodiments, the PEGylated protein contains PEGylated amino acid residues at least at K16, K32, K38, K40, K47, K67, K74, K82, K87, K88, K152, K154, K171, K312, and K321. In some embodiments, the PEGylated protein does not contain PEGylated 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. Cobalt (Co) is used at the active site of arginase 1. 2+ ) to replace natural manganese (Mn) 2+ It can enhance stability and catalytic activity at physiological pH. Polyethylene glycolation also prolongs the cycling half-life (t) of recombinant arginase 1. 1 / 2 ).

[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, homogenate clarification, SP Sepharose FF cation exchange capture chromatography, cobalt exchange, ultrafiltration / percolation, QSepharose FF anion exchange flow chromatography, Capto MMC multimode chromatography, and ultrafiltration / percolation. The purified Co-arginase 1 intermediate can be processed to form a PEGylated active pharmaceutical ingredient (API) or frozen and stored for later conversion to an API.

[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 includes loading a 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 the second protein product onto a multimode chromatography (MMC) column that captures arginase 1 and then elutes to provide a third protein product (“Third Protein Product”). In some embodiments, the third column (also referred to as “Column 3”) may 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, the first protein product, the second protein product, the 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] Cobalt loading of arginase 1 can be performed using various cobalt-containing solutions and at various temperatures. In one or more embodiments, the cobalt salt comprises Co. 2+ Such as CoCl2. In a preferred embodiment, cobalt loading of arginase 1 is carried out with CoCl2 at or around room temperature, such as at about 15 to about 25°C. Or about 20 to about The reaction is carried out at 25°C. The cobalt loading rate can be controlled by increasing or decreasing the reaction temperature. Cobalt loading can also be carried out within a certain pH range.

[0023] One aspect of this disclosure relates to altering conditions associated with CEX chromatography (column 1). The amount of protein loaded onto column 1 can be increased or decreased to select different arginase 1 charge variants. 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 per liter of CEX column resin volume) can produce arginase 1 with high specific activity. In various embodiments, loading factors are up to about 10 g / L, about 20 g / L, about 30 g / L, about 40 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 lysates 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 instead of MMC columns, such as SEC columns.

[0025] One aspect of the present invention relates to a method for producing recombinant cobalt-substituted human arginase protein (Co-rhARG). 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 cobalt salt to form cobalt-substituted recombinant human arginase protein (Co-rhARG), applying the cobalt-substituted recombinant human arginase protein (Co-rhARG) onto an anion exchange column and collecting the flow-through, adding the flow-through to a third chromatographic column, eluting the cobalt-substituted recombinant human arginase protein (Co-rhARG) from an MMC column with a high-salt solution, reacting with an 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, K87, 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: 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.

[0033] In one or more embodiments, the protein contains 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 exogenous protein fragments.

[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 exhibited a response to arginine hydrolysis at pH 7.4 at approximately 200 mM. -1 s -1 Approximately 4,000 mM -1 s -1 k within the range cat / K m .

[0039] In one or more embodiments, when measured in vitro, the protein exhibited 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 .

[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%, 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%.

[0044] In one or more embodiments, 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%.

[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] Treatment methods for arginase 1 deficiency

[0047] Another aspect of the invention relates to the administration of recombinant human arginase 1, such as Co-rhARG1-PEG. This 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 a dose of 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 this algorithm, the dosage 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 prior to this sample were a) the same dose level in mg / kg and b) consecutive (without missing doses).

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

[0052] Attached Figure Description

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

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

[0055] Figure 2 is a schematic diagram of an exemplary process of Escherichia 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 illustrates the column chromatography purification of arginase 1. Figure 4(a) shows loading E. coli cell lysates 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 lysates were applied to the column, followed by washing with approximately 1.5 L of buffer and elution 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 capturing arginase 1 onto a Capto multimode cation exchange column (column 3) and eluting with a high-salt solution to provide the 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) shows a representative chromatogram of the charge heterogeneous arginase 1. After 10–20 min, the charge variants of arginase 1 eluted from the analytical HPLC column. Figure 5(b) shows the same chromatogram as Figure 5(a), but with a higher peak magnification. Figure 5(c) shows the peak numbers assigned to the arginase 1 cation exchange charge variants. 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 LC / MS methods used to identify glucosylated arginase 1 variants produced by expressing rhARG in *E. coli*. LC / MS analysis identified unmodified arginase 1 (monomer), glucosylated arginase 1, phosphoglucosylated arginase 1, and 2X glucosylated arginase 1. Traces were obtained from two independent production runs of the pharmaceutical intermediate. Mass spectra were superimposed on RP LCMS at 35 °C for 33–35 min and the spectra were normalized to the peak intensity of the signal produced by unmodified arginase 1. The peak intensity of the variants was proportional to their relative abundance.

[0060] Figure 7 shows the results of applying a 0.0–0.2 M NaCl gradient to column 1. Fractions were collected at 0.25 CV (column volume) per 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 different gluconic acidified substances while maintaining product recovery.

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

[0062] Figure 9 shows the 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. Linearity plot (a) and semi-logarithmic plot (b) are shown. Note that the first mean BQL concentration is plotted at half the LLOQ (0.125 µg / mL). The mean circulating drug concentration in all patients increased with increasing Co-rhARG1-PEG dose. 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) data from a Phase 1 / 2 open-label study to evaluate Co-rhARG1-PEG administration in patients with arginase 1 deficiency. Using the dose escalation cessation 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). Other patients in the trial were assigned various dose levels from Part 2 by applying the dose escalation cessation criteria. These same criteria can be used to adjust (increase or decrease) the dose in any patient already receiving 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 was between 40 μM and 115 μM (dashed line). Subcutaneous administration of Co-rhARG1-PEG resulted in a longer duration of arginine concentration within this preferred range than 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. Figures are shown as averages of patient values, and data are derived from doses determined for each patient's discontinuation criteria.

[0065] Figure 12 shows plasma arginine and guanidino 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-L-arginine (NAA), α-keto-δ-guanidinopentanoic acid (GVA), and arginine (ARGA) at baseline and during OLE.

[0066] Figure 13 illustrates baseline deficits and clinical response outcomes. Figure 13(a) shows baseline deficits in patients with arginase 1 deficiency using the 6-minute walk test (6MWT), gross motor function measurement (GMFM) parts D and E, and the adaptive behavior assessment system (ABAS). Figure 13(b) shows clinical responses to the 6MWT, GMFM-D, and GMFM-E.

[0067] Figure 14 illustrates the time-dependent improvements of 6MWT, GMFM-D, and GMFM-E. Figure 14(a) shows the percentage of clinical responders for 6MWT and the percentage of patients with baseline deficiencies in 6MWT at doses 8 and 20. Figure 14(b) shows the percentage of clinical responders for GMFM-D and the percentage of patients with baseline deficiencies in 6MWT at doses 8 and 20. Figure 14(c) shows the percentage of clinical responders for GMFM-E and the percentage of patients with baseline deficiencies in 6MWT at doses 8 and 20.

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

[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 possesses enzymatic activity but is not very stable. Cobalt (Co) is used at the active site of arginase 1. 2+ ) to replace natural manganese (Mn) 2+ This method enhances 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 a pharmaceutical ingredient. In one or more embodiments, the pharmaceutical ingredient is polyethylene glycol-modified Co-arginase 1 (Co-rhARG1-PEG). Polyethylene glycolization of Co-arginase 1 significantly prolongs the cycling half-life. Similarly, although this document specifically refers to rhARG1, the methods, formulations, and uses described herein are also applicable 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 at least some of the rhARG1 in the natural manganese cofactors that are replaced by cobalt. In one or more embodiments, Co-rhARG1 is a separable intermediate in the production and / or purification process of Co-rhARG1-PEG.

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

[0074] The amount of Co-rhARG1-PEG active pharmaceutical ingredient can be expressed as the mass of the unpolyglycolated 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 *E. coli*. The hArg1 protein sequence was obtained from the NCBI database (UniProtKB: locus ARGI1_HUMAN, accession number P05089). Arginase 1 DNA was generated using overlapping oligonucleotides in a PCR reaction and codon-optimized for expression in *E. coli* (Figure 1(b)). The 321-amino acid arginase 1 monomer expressed in *E. coli* lacks the N-terminal methionine found in the native human arginase 1 monomer. The calculated molecular weight of Co-arginase 1 is 34721.6 Daltons (Table 1). The calculated molecular weight of homotrimeric Co-arginase 1 is 104164.8 Daltons. Arginase 1 contains no disulfide bonds.

[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 is 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 amount of PEG 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, 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 molecular weight (MW) of the PEG is about 5,000 Daltons.

[0079] In one or more embodiments, Co-rhARG1-PEG contains polyethylene glycol arginase. Polyethylene glycol arginase has the following two chemical names:

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

[0081] b.Des-Met 1 -Arginase-1 (hepatic arginase, EC 3.5.3.1) (Homo sapiens), in which manganese has been replaced by cobalt, and has an average of 10 primary amines (N-terminal serine and N-terminal arginine). 6 -Lysine) is amidated with [methoxypoly(vinyloxy)]acetyl, forming a non-covalent homotrimer.

[0082] Produced in Escherichia coli. The molecular formula of polyethylene glycol arginase is C636-C ... 1554 H 2492 N 416 O 453 S6[C3H4O2(C2H4O) n ] a Monomer. The average molecular weight of the trimer polyethylene glycol arginase is 284 kDa. The CAS registration number for polyethylene glycol arginase is 1659310-95-8.

[0083] The potential PEGylation sites of PEGylated arginase are shown below:

[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, as well as 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 PEGylation 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 PEGylation 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, the polyethylene glycol (PEG) reaction is performed by atomizing PEG and Co-rhARG1 for approximately 5 to 300 minutes, approximately 10 minutes to 300 minutes, approximately 20 minutes to 300 minutes, approximately 30 minutes to 300 minutes, approximately 5 minutes to 280 minutes, approximately 10 minutes to 280 minutes, approximately 20 minutes to 280 minutes, approximately 30 minutes to 280 minutes, approximately 5 minutes to 260 minutes, approximately 10 minutes to 260 minutes, approximately 20 minutes to 260 minutes, approximately 30 minutes to 260 minutes, approximately 5 minutes to 240 minutes, approximately 10 minutes to 240 minutes, approximately 20 minutes to 240 minutes, and approximately 30 minutes 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 the 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 PEGylated in the range of about 15% to about 60%. In one or more embodiments of Co-rhARG1-PEG, 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, K82 is PEGylated in the range of about 30% to about 98%. In one or more embodiments of Co-rhARG1-PEG, K87 is PEGylated in the range of about 15% to about 65%. In one or more embodiments of Co-rhARG1-PEG, K88 is PEGylated in the range of about 25% to about 70%. In one or more embodiments of Co-rhARG1-PEG, K152 is PEGylated in the range of about 25% to about 85%. In one or more embodiments of Co-rhARG1-PEG, K154 is PEGylated in the range of about 15% to about 65%. In one or more embodiments of Co-rhARG1-PEG, K171 is PEGylated 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 polyethylene glycolation. In one or more embodiments, about 1 to about 20 moles of PEG have PEGylated 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 was measured to demonstrate PEG scavenging and stability. In some embodiments, the free PEG concentration (μg) in polyethylene glycolated Co-rhARG1 (mL) was 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, the conversion of L-arginine to L-ornithine and urea. The PEGylated active pharmaceutical ingredient Co-rhARG1-PEG catalyzes the same reaction. Enzyme activity was assessed by measuring the L-arginine to L-ornithine conversion at pH 7.4 and 37°C for a fixed reaction time. The conversion of products was converted to a reaction rate and fitted to the Michaelis-Menten equation to determine K. m and k cat .

[0091]

[0092] V max The maximum reaction rate reached at saturated substrate concentration; K m It is the Michaelis-Menten binding constant, used to measure V. max The substrate concentration at half the rate. Enzyme turnover number k. cat By V max / [E] Calculation.

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

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

[0095] In one or more embodiments, the protein (e.g., Co-rhARG1 or Co-rhARG1-PEG) exhibited a concentration greater than 200 mM at pH 7.4. -1 s -1 k cat / K M In a specific embodiment, the protein exhibited a concentration of approximately 200 mM at pH 7.4. -1 s -1 Approximately 4,000 mM -1 s -1 k within the range cat / K M In another embodiment, the protein at pH 7.4 and 37°C exhibited a concentration of approximately 400 mM. -1 s -1 Approximately 2,500 mM -1 s -1 k within the range cat / K M 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 concentration greater than 400 mM at 37°C and pH 7.4. -1 s -1 k cat / K M Example k cat / K M Values ​​included at pH 7.4 and 37°C: approximately 200, approximately 250, approximately 300, approximately 350, approximately 400, approximately 450, approximately 500, approximately 550, approximately 600, approximately 650, approximately 700, approximately 800, approximately 900, approximately 1,000, approximately 1,100, approximately 1,200, approximately 1,500, approximately 2,000, approximately 2,500, approximately 3,000, approximately 3,500, and approximately 4,000 mM. -1 s-1 , or any range between these values.

[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 98%, 98.5%, 99%, or 99.5% identity with SEQ ID NO: 1. 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 comprises 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 comprises 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. This change in cobalt cofactor leads to a change in the K+ of arginine at pH 7.4. m The concentration varies 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 removal and stability. In some embodiments, free cobalt was 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 protein efficacy and is an indicator of bound cobalt, as 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 specific embodiment, the Co-rhARG1-PEG active pharmaceutical ingredient contains about 2 μg Co / mg protein and about 0.05 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] Shaking bottle expansion

[0107] The purpose of shake-flask expansion / fermentation is to generate inoculum, which is then used to inoculate the production fermenter. Shake-flask expansion produces cell clusters for inoculating the production reactor, as well as 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 the second inoculation.

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

[0110] Fermentation production

[0111] The purpose of production fermentation is to scale up 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. Post-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, jetting 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 cooling, the sterilized post-additions kanamycin, glucose, and potassium phosphate are sterilized and filtered into the production fermenter. If necessary, the sterile medium is mixed with purified water to the specified pre-inoculation weight using a 0.2 µm sterile filter. The fermentation medium is titrated to a controlled pH with an alkali (ammonium hydroxide).

[0113] Aseptic inoculation of the production fermenter at 37°C was performed using a combined inoculum via pressure-assisted transfer. Fermentation broth samples were collected and measured at fixed frequencies for OD600 analysis from inoculation time to fermentation cooling. Glucose samples were collected at fixed intervals starting 3 hours post-inoculation, with the frequency increased at 9 hours post-inoculation. Antifoaming solution was added as needed during fermentation to prevent 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 was preferably maintained at 36–38°C and a pH of 7.0–7.4, with agitation and aeration.

[0114] The nutrient feed consisted of yeast extract, Martone B-1, L-cysteine ​​hydrochloride, and glycerol. Feeding began when the glucose concentration was less than 10 g / L (12–14 hours post-inoculation) and continued at a fixed rate until the end of production. Expression was triggered by the addition of IPTG. Induction lasted 18 hours. After fermentation, the mixture was cooled in preparation for harvesting. The production fermenter produced approximately 6 g / L of soluble arginase 1 titer. An overview of the production fermentation is shown in Figure 3.

[0115] Harvesting Operation

[0116] The harvesting operation captures cells containing soluble arginase 1, breaks down / lyses the cells, and removes cell debris 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 cell slurry is resuspended in 25 mM HEPES at pH 7.6 and then homogenized twice. Alternatively, a pH range of 7.2–7.6 for 25 mM HEPES can be used. The lysed material is clarified using a centrifuge to remove cell debris and then 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, high pressure is used for cell disruption. The cell slurry is transferred to a homogenizer at a controlled rate, and the homogenized effluent is passed through a heat exchanger to reduce temperature rise during pressure homogenization. The frozen cells undergo two homogenization processes. The merged product from 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 then transferred at a controlled rate to a disc-type batch discharge centrifuge. The clarified lysate is collected for further processing.

[0120] Filter the clarified lysate, such as using a filter of approximately 0.2 µm. A process transfer filter can also be used for microbial control during process operations. For this purpose, the filter can be either 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 used before each downstream process step to mitigate potential bioburden.

[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. The 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 more embodiments, the CEX column is the first column (“Column 1”) in a system with 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 is bound at salt-free or low salt concentrations. In one or more embodiments, rhARG1 is eluted with a buffer having a high salt 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, followed by elution 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 instead of the Mn cofactor. In one or more embodiments, Co is used. 2+ Co loading is achieved using salts such as CoCl2. The incubation time is temperature-dependent; therefore, lower cobalt substitution temperatures require longer incubation times, while higher cobalt substitution temperatures do not require longer incubation times. Cobalt loading temperatures can range from as low as 1°C to above 50°C, with corresponding incubation times ranging from over 8 hours to less than 10 minutes.

[0128] Various embodiments provide the reaction of rhARG1 or Co-rhARG1 with a polyethylene glycolation reactant such as methoxyPEG succinimide carboxymethyl ester (MW 5000). The polyethylene glycolation reactant is typically provided in a 10-40 molar excess compared to the enzyme. Incubation time can range from 0.5 to 4 hours. The pH during polyethylene glycolation can be from about 8 to about 9, such as 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 contains 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% phosphoglucosylated arginase I. Exemplary amounts include about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, or about 9% of phosphorylated 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% phosphorylated arginase I.

[0131] Due to the level of heterogeneity in PEGylated trimers, imaging capillary isoelectric focusing (iCIEF) provides a measurement of the homogeneity of PEGylated proteins. In one or more embodiments, iCIEF analysis of purified PEGylated proteins 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 the proportion of that particular substance. In one or more embodiments, certain peaks may be combined 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 of 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 of 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 or Co-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 of peak 7 in proportions of 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 area under the curve of peak 8 in a proportion of 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 area under the curve of peak 9 that is 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] Application of rhARG1, Co-rhARG1 and Co-rhARG1-PEG

[0142] rhARG1, Co-rhARG1, and Co-rhARG1-PEG (and compositions comprising them) as described herein may be administered via 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).

[0143] Compositions containing rhARG1, Co-rhARG1, and Co-rhARG1-PEG can be provided in formulation form along with physiologically tolerable liquid, gel, or solid carriers, diluents, and excipients. Such compositions are typically prepared as liquid solutions or suspensions for injection. Suitable diluents and excipients include, for example, water, saline, glucose, glycerol, and combinations thereof. Furthermore, if desired, the composition may contain small amounts of auxiliary substances 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] Baseline plasma arginine concentrations should be obtained before initiating treatment. The recommended initial dose of PEGylated arginase for ARG1-D patients is 0.10 mg / kg, administered once weekly as a single intravenous infusion. If the initial dose of 0.10 mg / kg fails to reduce plasma arginine to ≤150 μmol / L, the dose can be adjusted to a maximum of 0.20 mg / kg, once weekly. If plasma arginine levels decrease to below 50 μmol / L during treatment, dose reduction should be considered. After five or more IV doses of PEGylated arginase, subcutaneous administration of PEGylated arginase should be considered for ARG1-D patients, with continued regular monitoring of plasma arginine levels.

[0147] Recommended subcutaneous administration regimen:

[0148] When transitioning from intravenous to subcutaneous administration of PEGylated arginase, administer the first subcutaneous dose, not the planned next intravenous dose. The initial subcutaneous dose should be the same as the mg / kg dose administered at 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] After initiating treatment with pegylated arginase, plasma arginine monitoring should be performed 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 application instructions

[0152] Polyethylene glycol arginine enzyme is supplied as a frozen liquid formulation in 10 mL single-use glass vials containing 5 mL of PEG arginine enzyme at a concentration of 1 mg / mL or 5 mg / mL. Each single-use glass vial of PEG arginine enzyme is intended for single intravenous or subcutaneous injection. Visually inspect the PEG arginine enzyme for particulate matter and discoloration before administration. PEG arginine enzyme is a colorless to pale yellow or pale pink solution. Discard the vial if it is discolored, cloudy, or contains particulate matter. Remove the cap from the vial. Sterilize the vial's rubber 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 required, draw the solution from each vial using a separate needle. Calculate the amount of drug to be drawn from the vial for use in the infusion pump. After drawing the appropriate volume of drug into the syringe, draw physiological saline using a separate needle to achieve a total volume of 40 mL. Calculate the required amount of drug as follows:

[0153]

[0154] PEGylated arginase was administered via intravenous infusion over a 30-minute period using a syringe pump.

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

[0156]

[0157] In one or more embodiments, the volume used 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 in 10 mL vials, as described below:

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

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

[0162] Warnings and precautions

[0163] Hypersensitivity reactions may occur with the use of PEG arginine enzyme. Monitor all patients for signs and symptoms of acute allergic reactions (e.g., urticaria, pruritus, erythema, hypotension, tachycardia) during and after PEG arginine enzyme infusion. In the event of a severe hypersensitivity reaction, immediately slow or discontinue PEG arginine enzyme administration and provide 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 PEGylated arginase causes harm to the fetus. However, there are no adequately controlled studies in pregnant women. Because animal reproductive studies are not always predictive of human responses, PEGylated arginase should only be used during pregnancy when clearly necessary.

[0166] Breastfeeding women

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

[0168] illustrate

[0169] Polyethylene glycol arginase is a cobalt-substituted recombinant human arginase I enzyme covalently conjugated with monomethoxy polyethylene glycol (mPEG). It 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 polyethylene glycol arginase, the manganese cofactor is replaced by cobalt to produce Co-arginase I. Replacing native manganese (Mn+2) with cobalt (Co+2) at the active site of arginase I enhances stability and catalytic activity at physiological pH. PEGylation 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 of arginine to ornithine per minute at 37°C.

[0170] Polyethylene glycol arginine enzyme is intended for intravenous or subcutaneous infusion and is provided as a sterile, clear, colorless to pale yellow or pale pink solution, prepared at 1 mg / mL and 5 mg / mL concentrations in a buffer solution containing 50 mM sodium chloride, 5 mM potassium phosphate, and 1.5% w / v glycerol at pH 7.4. It is provided as a preservative-free, sterile solution in single-use clear glass vials. Each 1 mg / mL vial of finished polyethylene glycol arginine enzyme contains 5 mL of finished product (5 mg of polyethylene glycol arginine enzyme per vial). Each 5 mg / mL vial of finished polyethylene glycol arginine enzyme contains 5 mL of finished product (25 mg of polyethylene glycol arginine enzyme 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] Following treatment with polyethylene glycol arginase in adult and pediatric patients with arginase 1 deficiency, serum arginine concentrations decreased from baseline to the normal serum arginine range of 40 to 115 μmol / L. Maximum inhibition of L-arginine was observed approximately 8 hours post-administration, decreasing in a dose-dependent manner and returning to pre-administration levels within 168 hours. A strong correlation was observed between polyethylene glycol arginase and arginine, with immediate inhibition of arginine upon IV administration and a maximum reduction in arginine concentrations reaching within 24 hours post-administration.

[0173] Pharmacokinetics

[0174] Pharmacokinetic samples were collected throughout the dosing interval (0–168 hours) following intravenous administration to characterize the relationship between polyethylene glycol arginase pharmacokinetics and arginine. Pharmacokinetics were assessed via Cp throughout the entire dose range (0.015 mg / kg to 0.2 mg / kg). max and AUC 0-168 Measured PEGylated arginase exposure increased approximately proportionally to the dose, with a 13-fold increase in dose resulting in C... max and AUC 0-168 Increased 14-fold. No accumulation of polyethylene glycol arginine enzyme was observed following a once-weekly IV dosing regimen, and T values ​​remained within the entire dose range. 1 / 2 The exposure duration was approximately 30 hours, and it exhibited 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 total kinematic energy (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 administration 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 male reproductive organ weight and adverse sperm analysis results. At 1.0 mg / kg, adverse effects on sperm analysis were observed, namely 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 both 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, given that the normal sperm development cycle is approximately 9 weeks or longer than a 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 men, based on microscopic changes in the testes at 0.3 mg / kg and 1.0 mg / kg, the NOAEL was 0.1 mg / kg.

[0179] Cynomolgus monkeys were administered 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. Potential toxicity and total kinematic kinase (TK) of PEGylated arginine enzyme 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 observed in clinicopathological parameters (coagulation, growth hormone, and urinalysis), ECG and ophthalmological examination, respiratory rate, and blood pressure assessment.

[0180] How to provide / store and process

[0181] Polyethylene glycol arginase is provided as an injection.

[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

[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 disclosures, the following abbreviations are used: eq (equivalent); M (molar concentration); µM (micromolar concentration); mM (millimolecular concentration); N (normal); mol (molar); mmol (millimole); µmol (micromolar); nmol (nanomolar); g (gram); mg (milligram); µg (microgram); L (liter); ml (milliliter); µl (microliter); cm (centimeter); mm (millimeter); µm (micrometer); nm (nanometer); MW (molecular weight); PBS (phosphate-buffered saline); min (minute).

[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-associated and process-associated 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 binds arginase 1 at pH 7.6 in the absence of salt using cation exchange chromatography and elutes with a buffer of increasing salt concentration (Figure 4(a)). In one embodiment, the salt is NaCl, and elution of column 1 is 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] Figure 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 significant 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 (the final peak) was eluted with 0.1 M NaCl, detected by absorbance 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 cobalt concentrations (see Table 2). Incubation times for cobalt substitution can be as short as 10 minutes and carried out at temperatures above 50°C. Conversely, cobalt loading temperatures 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 the Column 1 composite) can be maintained at room temperature for use in the cobalt substitution step. In one embodiment, the stock solution of cobalt chloride (0.5 M CoCl2) is diluted 50-fold by adding it to the Column 1 composite at a prescribed rate, resulting in a final cobalt chloride concentration of 10 mM. The cobalt substitution reaction mixture is then mixed at 20°C for two hours. In another embodiment, cobalt loading of arginase 1 is 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

[0194]

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

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

[0197] The UF / DF1 membrane was cleaned using the following steps: a 2 M NaCl rinse was performed, followed by a denaturing cleaning step with 0.5 N NaOH, and then circulated for 30 minutes. The system was rinsed with purified water, and the NWP (Natural Power Plant) was tested to evaluate the effectiveness of the cleaning procedure. The membrane can 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 chromatography (column 2)

[0200] A preferred embodiment of arginase 1 purification uses another column (“Column 2”) as an anion exchange chromatography column. 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 confluent, such as host cell DNA and endotoxins. Column 2 binds these impurities, while Co-arginase 1 flows through and is collected in the column effluent during loading and washing steps. In one embodiment, anion exchange flow 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, the first protein product is loaded onto an anion exchange column to capture impurities while arginase 1 is recovered from the flow-through. Figure 4(b) is a representative chromatogram of arginase 1 purified on an anion exchange column (column 2). The absorbance at 280 nm indicates a significant amount of protein 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.

[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 embodiments using MMC, arginase 1 is captured on the column, while process-related impurities such as host cell proteins (HCP), DNA, and endotoxins are eluted in the flow. In this embodiment, Co-arginase 1 can be captured by the column at pH 8.4 in the absence of salt, followed by elution with a buffer of 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 of resin and elutes using a high-salt step with 50 mM Tris, 250 mM NaCl, and pH 8.1–8.5. In several embodiments, flow from the anion exchange column (column 2) is 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 bound Co-arginase 1.

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

[0206] UF / DF 2 concentrates arginase 1 and exchanges the protein into a pre-polyglycolated 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 perform buffer exchange on the column 3 consumable of the unpolyglycolated Co-arginase 1 intermediate before PEGylation (or before additional filtration and storage). 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 consumable (also known as the third protein product) is perfused five times its percolation volume against 20 mM sodium phosphate, 50 mM sodium chloride, 1.5% (w / v) glycerol, pH 7.4. If the column 3 consumable concentration is < 8 g / L, the consumable is further concentrated to 8 g / L. After percolation (and concentration, if necessary), the combined solution is recycled and recovered from the system using twice the system retrieval volume of 20 mM sodium phosphate, 50 mM sodium chloride, and 1.5% (w / v) glycerol at pH 7.4. Following recovery, a two-step dilution can be performed using the percolation solution. The target concentration for the first dilution is 6 g / L, and the target concentration for the second dilution is 5 g / L. Both steps can be used to achieve the target concentration. If the concentration after the first dilution is within the target range, the second step may not be necessary.

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

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

[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 merged product (fresh or thawed) to provide conditions optimal 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 normalized permeability test (NWP) is performed, and the membrane is then equilibrated before use in production. Once the UF / DF system is equilibrated, 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 merged product is concentrated and recycled from the system using twice the system retentate 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. Use a two-step process 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 the UF / DF 2 and UF / DF 3 steps, the first buffer exchange can be performed in 20 mM sodium phosphate, 50 mM NaCl, 1.5% glycerol, pH 7.4, ≥ 5 DV, with the protein concentrated to approximately 5.0 mg / mL. The second buffer exchange can be performed in 0.1 M sodium phosphate, pH 8.1-8.5, with the protein concentrated to approximately 10.0 mg / mL (in preparation for the polyethylene glycolation of the active pharmaceutical ingredient).

[0211] Example 8: Polyethylene glycolation of arginase 1

[0212] PEGylation covalently links PEG (polyethylene glycol) to the Co-arginase 1 (API) molecule (see Table 4 for representative examples of the PEGylation step). In one embodiment, the PEGylation reaction covalently binds a 5000 Da PEG molecule 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 a solid or liquid methoxyPEG succinimidyl carboxymethyl ester, which reacts with lysine residues on Co-arginase 1. The resulting PEGylated protein (Co-rhARG1-PEG) has a molecular weight of approximately 280 kDa. The PEGylated conjugate can be filtered and stored at 2–8°C until UF / DF4 operation.

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

[0214]

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

[0216] Following polyethylene glycolation, unbound PEG is removed by ultrafiltration / percolation, arginase 1 is exchanged into the formulation buffer, and arginase 1 is concentrated for use in 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 is equilibrated, the PEG conjugate is percolated ten times the dialysate volume against 5 mM potassium phosphate, 50 mM sodium chloride, 1.5% (w / v) glycerol, pH 7.4. 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, arginase 1 was exchanged in 20 mM sodium phosphate, 50 mM NaCl, 1.5% glycerol, pH 7.4, and adjusted to a protein concentration of approximately 5.0 mg / mL.

[0217] In some embodiments, it has been found that the formulation buffer of 5 mM potassium phosphate, 50 mM sodium chloride, 1.5% glycerol, pH 7.4 enhances the stability of arginase 1 during storage compared to other buffers such as sodium phosphate buffer. In one or more embodiments, the 5 mM potassium phosphate buffer comprises 1 mM K₂HPO₄ and 4 mM KH₂PO₄.

[0218] The active pharmaceutical ingredient (Co-rhARG1-PEG) is a polyethylene glycol-modified cobalt-substituted human arginase 1, prepared by conjugating activated PEG molecules to 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 per protein using phthalaldehyde. Phthalate reacts specifically with primary amines in the presence of thiols to form fluorescent derivatives. Measurement of the fluorescence signal allows for quantification of the active free amines present in the 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 active pharmaceutical ingredient was measured by SEC-HPLC and detected by refractive index. 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 API

[0220]

[0221] Example 9: CIEX-HPLC Characterization of Pharmaceutical Intermediates

[0222] During E. coli fermentation, various arginase 1 charge variants may be generated. These 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 the mobile phase gradient according to Table 6.

[0223] Table 6: CIEX-HPLC Gradient Program for Charge Variant Co-arginine 1 Intermediate

[0224]

[0225] Samples were diluted with formulation buffer prior to analysis. Results are described as percentage charge variant distributions. 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 active pharmaceutical ingredient

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

[0228] In the presence of an applied electric field, analytes in iCIEF (i-capillary isoelectric focusing) migrate through the capillary via the counter-current migration of hydrated hydrogen ions (anolyte) and hydroxide ions (cathode electrolyte). Samples are diluted in a matrix containing a carrier ampholyte 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. Ultraviolet absorption images of the entire capillary are digitally captured every 30 seconds and after the completion of each focusing step.

[0229] The results can be expressed as a percentage of charge variation distribution. Figure 5(b) shows a representative electrophoresis plot where nine major peaks of the active pharmaceutical ingredient are observed. Peaks 3 and 4 are grouped 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]

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

[0233] An enzymatic assay for measuring the activity and identifying the 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 ranging from 0 to 2 mM. The reaction is performed at 37 °C for a fixed time. Reaction times have 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] Figure 8(a) (Co-arginase 1 intermediate) and Figure 8(b) (Co-rhARG1-PEG active pharmaceutical ingredient) show examples of reaction rate versus substrate concentration plots and representative K values.cat K m and K cat / K m .

[0235] Example 12: 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, thus measuring 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 active pharmaceutical ingredient

[0240]

[0241] Example 13: Post-translational modification of Co-arginase 1 intermediate

[0242] Post-translational modifications of the Co-arginase 1 intermediate were detected using a variety of techniques, such as peptide mapping, intact LC-MS, and reversed-phase LC / MS. Table 10 summarizes all identified modifications.

[0243] Table 10: Modifications of identified Co-arginase 1 intermediates

[0244]

[0245] Characterization determined that the primary modification in the presence of Co-arginase 1 intermediates was N-terminal gluconication (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). Analytical methods typically require dissociation of arginase into monomers and analysis as monomers. N-terminal gluconication of arginase 1 (analyzed as a monomer) was typically 10.8% to 13.9%. Other modifications in samples from the three time points were N-terminal phosphogluconic acid-modified monomers (4.3% to 6.5%) and digluconic acid-modified monomers (0.7% to 1.2%). In samples from standardized reference production runs, the levels of unmodified Co-arginase 1 (monomers) and Co-arginase 1 intermediates were comparable, ranging from 80.6% to 83.6%. The standard conditions used for the purification process (i.e., column 1 without salt gradient) 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 the conditions of column 1

[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 illustrates the 0.0 to 0.2 M NaCl gradient applied to column 1. The fractions collected from the eluent of column 1 were analyzed by SE-HPLC, CEX-HPLC, and RP-HPLC.

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

[0251] Alternative NaCl gradients can be used for column 1 elution, such as 0 to 0.5 M NaCl. 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 active pharmaceutical ingredients or finished products.

[0252] Further analysis of the first protein product (and arginase 1 variant) was performed by LC / MS (see Figure 6). LC / MS analysis identified the specific types 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 2-fold (2X) glucosylated arginase 1.

[0253] Table 12 shows the fractions with different gluconic acidification 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 show that the main peak (peak 5) has a high percentage of ungluconic acidified 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 properties.

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

[0255]

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

[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.

[0258] Example 15: Changes in fermentation conditions

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

[0260] Table 13: Glutamate-enhanced arginase 1 observed in fermentation vessels

[0261]

[0262] Example 16: Changes in the loading factor of column 1

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

[0264] Table 14: Effect of Column 1 Loading Factors on Protein Product 1

[0265]

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

[0267] The finished drug product manufactured using the method of this invention was used in a Phase 1 / 2 open-label study to evaluate the administration 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. Secondary endpoints included: determining the effect of IV administration of the study drug on plasma arginine concentrations; determining the effect of IV administration of the study drug on plasma guanidine compounds (GC); and characterizing the pharmacokinetic (PK) profile of IV administration of the study drug. 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 lowering plasma arginine levels. Furthermore, control of plasma arginine levels was accompanied by clinically significant responses in activity and adaptive behavior. Treatment was generally well tolerated. Hypersensitivity reactions were rare and could be managed with standard interventions.

[0269] The Co-rhARG1-PEG finished product provided for the study was a liquid formulation packaged in a 10 mL disposable glass vial, containing 5 mL of the prepared finished product at a concentration of 1 mg / mL. The drug was prepared 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 for this Phase 1 / 2 trial, including the open-label extensions 101A and 102A, is shown in the figure below:

[0271]

[0272] Part 1 involves the patient trying the drug and focuses on safety. Part 2 is designed to allow patients to receive a consistent dose and to identify biomarkers of clinical efficacy. Baseline assessment of arginine levels is performed before each part. All patients who participated in Part 1 and 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, at 2-week intervals as needed to optimize plasma arginine levels. 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: a patient's plasma arginine level < 40 μM for all samples collected over a period of at least 40 (± 2) hours post-dose administration, or a patient's mean plasma arginine level < 115 μM for all samples collected over a period of at least 112 (± 2) hours post-dose administration.

[0274] If none of these events occur, 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 be increased beyond 0.30 mg / kg for therapeutic purposes.

[0275] Part 2 covers the repeated dosing period for patients who completed Part 1. Part 2 establishes a dosage and regimen for each patient that safely optimizes plasma arginine levels in the range of approximately 40 μM to approximately 115 μM during repeated dosing, with a focus on maintaining pre-dose levels below 150–200 μM. Multiple dosage levels may be used in Part 2 if data suggest the potential for better investigation of dose-response outcomes during repeated dosing. Arginine levels during treatment will also be compared to pre-treatment arginine levels.

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

[0277] result

[0278] Among all patients, the average C max and average AUC 0-168The increase was proportional to the dose. For Co-rhARG1-PEG dose levels of 0.015, 0.03, 0.06, 0.1, and 0.2 mg / kg, the mean (± SD) C max The values ​​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. There were differences in mean C between ADA-positive and ADA-negative patients. max Mild anti-drug antibody (ADA) effect (Figure 9).

[0279] Changes in AUC (AUC 0-168 AUC 0-∞ The dose was proportional to the dose within the studied dose range; note that there was no significant change 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) estimates 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, within the assessed dose range, there was a trend towards increasing mean circulating drug concentrations in all patients using escalating doses of Co-rhARG1-PEG. Following the first administration of Co-rhARG1-PEG in Part 2, the mean C... max The increase was proportional to the dose. 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)C max The values ​​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 Part 2, week 8, mean circulating drug concentrations generally increased with increasing doses of Co-rhARG1-PEG across all patients. At week 8, available PK concentrations had no significant ADA effect. 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, mean circulating drug concentrations (C) in all patients generally increased with increasing doses of Co-rhARG1-PEG. max and AUC 0-168 The increase is proportional to the dosage.

[0282] In addition to pharmacokinetic data, pharmacodynamic (arginine) data were also collected (Figure 10). In Part 2, patients with arginase 1 deficiency were administered Co-rhARG1-PEG at a QW IV dose (weekly), and the starting dose was selected based on the observed PD (arginine) response in Part 1. Following the first QW IV dose of Co-rhARG1-PEG, circulating arginine levels decreased significantly, particularly for doses equal to or greater than 0.04 mg / kg. In some cases, individual arginine concentrations dropped 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, Co-rhARG1-PEG exposure typically increases, and arginine inhibition increases with increasing dose. Individualized dose optimization was performed in Part 1, resulting in different numbers of patients at each dose level in Week 1 and Week 8 of Part 2.

[0284] Example 18: Subcutaneous application

[0285] After the completion of Part 2 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 presented pharmacodynamic characteristics that appeared to be superior to IV administration. Equally 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 maintains patients' arginine levels within the preferred (healthy) target range for 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 approximately 40 μM to approximately 115 μM (during repeated dose administration), with an emphasis on maintaining levels 150–200 μM lower than pre-administration levels. As shown in Figure 11, subcutaneous administration of Co-rhARG1-PEG results in arginine concentrations that are lower than 40 μM and higher than 115 μM. Surprisingly, subcutaneous administration yields 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 in Phase 1 / 2 Clinical Trials and Open-Label Extensions

[0288] Sixteen patients (11 children and 5 adults) were enrolled in Part 1 of 101A, and 15 patients progressed to 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] The patients’ 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) shows the plasma levels of guanidinoacetic acid (GAA), N-α-acetyl-L-arginine (NAA), α-keto-δ-guanidinopentanoic acid (GVA), and arginine (ARGA) at baseline, as well as the decrease in plasma GC levels during OLE.

[0293] Of the 16 patients, 15 completed all activity assessments at baseline (Patient 13 was wheelchair-bound) (Figure 13(a)). Defect was defined as: 6MWT: less than the 5th below. Percentiles; GMFMD: < 35 out of 39; GMFME: < 68 out of 72; ABAS-3: ≤ 85. 88% of patients (14 out 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 sections, respectively. Ten patients were eligible for the ABAS-3 assessment of adaptive behavior at baseline. Six patients were not tested due to 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 out of 14 patients (79%) were defined as responders at dose 20 (Figure 13(b)). The 20-dose data demonstrated 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 5 patients (100%) who reached dose 44 maintained their overall clinical response status at dose 20 as responders.

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

[0296] Data from all patients following 20 doses of PEGylated arginine enzyme (PEGArginase) demonstrated a significant and sustained reduction in plasma arginine, improved key disease outcomes, and a clinical response 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 PEGArginase. PEGArginase was well-tolerated, and the incidence of treatment-related adverse events decreased over time. Evidence of improved arginine control and clinical benefit following PEGArginase 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 underway using Co-rhARG1-PEG produced by the method of this invention to investigate the efficacy and safety of Co-rhARG1-PEG in children and adults with arginase 1 deficiency. This trial is currently ongoing as a study using polyethylene glycol arginase (PEG). P ) Influence( E Arginase 1 deficiency ( A Clinical C End point ( E ) or PEACE (CAEB1102-300A; NCT03921541).

[0299] The study design for this three-phase trial is shown in the figure below:

[0300]

[0301] Key inclusion criteria

[0302] a. Patients aged ≥ 2 years, diagnosed with ARG1-D, and with plasma arginine levels ≥ 250 μmol / L, were included in a statistical test to assess the proportion of patients achieving plasma arginine levels below the medically recommended value of 200 μmol / L.

[0303] b. Ability to maintain a stable and consistent diet during blind testing.

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

[0305] d. Able to perform and successfully complete clinical assessments, and must have baseline deficiencies on 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. Active infection within 3 weeks prior to receiving the first dose of pegylated arginase.

[0309] c. Extreme mobility impairment, defined as being unable to perform an assessment on the Gillette Functional Assessment Questionnaire (GFAQ) or having a GFAQ score of 1 (completely unable to complete any steps).

[0310] d. Has participated in previous studies on polyethylene glycol arginase intervention or is currently participating in other clinical trials.

[0311] e. History of polyethylene glycol allergy

[0312] Table 16: Definition of baseline defects in 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 Toolkit (U.S. Department of Health and Human Services, Washington, D.C.) Motion Domain Dataset (2-Minute Walking Endurance Test)

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

[0317] Secondary endpoint measures include:

[0318] a. Clinical response endpoint: A clinically responding patient was defined as a patient 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 of each individual assessment scale at the clinical response endpoint

[0320] c. Other clinical outcome assessments

[0321] i. Functional Activity Scale (5, 50, 500 meters)

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

[0323] iii. Vincent 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. Characterization of the pharmacokinetic properties of polyethylene glycol arginase

[0327] Table 17: Definition of Clinical Response Endpoints in Clinically Responding Individuals

[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 (3-4 week screening, 24 weeks treatment, followed by an 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 implemented by an informed pharmacist and / or physician according to the 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 investigator and sponsor approval. The initial mg / kg subcutaneous dose may be the same as the IV dose.

[0330] Subjects administered Co-rhARG1-PEG began at dose level 2 (see Table 18 below), i.e., 0.10 mg / kg. Starting from the 5th visit, the dose will be adjusted by an informed physician based on plasma arginine levels according to the following dosing algorithm, if necessary:

[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 a dose was missed), the dose is reduced by one dose level (see Table 17), but not 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 average of the last four plasma arginine measurements that met strict pre-specified criteria. After 24 weekly doses, the mean decrease in plasma arginine levels from baseline in patients treated with PEGylated arginase will be compared 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 difference of 200 μM in mean plasma arginine levels 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 the clinical response endpoint between groups 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 PEGylation analysis. Three Co-rhARG1-PEG batches were analyzed. Peptide mapping was performed as follows: Co-rhARG1-PEG active pharmaceutical ingredient, Co-Arginase L intermediate (non-PEGylated) batch, and corresponding reference standard 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. The resulting peptides were resolved using an acetonitrile gradient in 0.05% trifluoroacetic acid on a Waters Acquity ВЕН 300 C18 column, 2.1 × 150 mm, Waters C / N 186003687. LC-MS and MS / MS lysis of the peptides were obtained on a Waters Xevo G2-XS QTOFMS / MS.

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

[0342] Throughout this specification, references to "one embodiment," "some embodiments," "various embodiments," "one or more embodiments," or "embodiment" are intended to indicate that a particular feature, structure, material, or characteristic described in connection with that embodiment is included in at least one embodiment of this disclosure. Therefore, terms such as "in one or more embodiments," "in some embodiments," "in various embodiments," "in one embodiment," or "in an embodiment" appearing throughout this specification do not necessarily refer to the same embodiment of the invention. Furthermore, specific features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments.

[0343] While the disclosure herein provides for descriptions of 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.

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 Escherichia 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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