Methods for production of human recombinant arginase 1 and uses thereof

Abstract

This invention describes a method for producing recombinant arginase, such as polyethylene glycol-modified cobalt-substituted recombinant human arginase 1. It also describes pharmaceutical compositions comprising such recombinant arginase; methods of treatment; and uses of such recombinant arginase.

Description

Technical Field

[0001] This disclosure generally relates to enzyme replacement therapy and the treatment of arginase 1 deficiency or hyperargininemia. This disclosure also encompasses methods for producing human recombinant arginase 1. Arginase 1 can also be used to treat cancer. Prior Art

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

[0003] Arginase 1 is primarily found in red blood cells (RBCs) and the liver. ARG1 is the only gene in which mutations are known to 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 diplegia, seizures, and growth failure. If untreated, the disease progresses to severe spasticity, loss of ambulation, bowel and bladder incontinence, 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 mildly elevated urinary orotic acid. Most patients have undetectable arginase 1 enzyme activity in their RBCs (<1% of normal).

[0004] Current treatment for arginase 1 deficiency focuses on maintaining plasma arginine concentrations as close to normal as possible through lifelong dietary protein restriction. Protein intake is limited to the minimum required to maintain protein biosynthesis and growth. Half or more of the dietary protein is given as a mixture of essential amino acids that does not contain arginine. This dietary modification can reduce plasma arginine levels in most patients, but the diet is unpalatable, expensive, and difficult to maintain and manage, especially in growing children.

[0005] Patients with arginase 1 deficiency have limited treatment options, highlighting the significant unmet need for therapies that reduce arginine levels to within the normal range and promote lifelong maintenance of normal arginine levels. Development of such a therapy could be used to minimize exposure to the neurotoxic effects of arginine and its metabolites and provide these patients with the potential for normal neurocognitive development.

[0006] In addition to treating arginase 1 deficiency or hyperargininemia, 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 agents such as pembrolizumab. Summary of the Invention

[0007] [Production of arginase]

[0008] One aspect of the present invention relates to methods for producing and / or purifying recombinant human arginase proteins. 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 rhARG1 is specifically mentioned herein, the methods, formulations, and uses described herein are also applicable to rhARG2.

[0009] In certain embodiments, the production of an arginase drug substance comprises the following key steps: fermenting an E. coli strain 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 the drug substance (Co-rhARG1-PEG). In one or more embodiments, Co-rhARG1-PEG comprises pegylated arginase.

[0010] One or more embodiments of Co-rhARG1-PEG involve cobalt-substituted, PEGylated human recombinant arginase 1 expressed in E. coli and formulated for intravenous (IV) or subcutaneous (SC) administration. The use of cobalt (Co 2+ ) in place of native manganese (Mn 2+ ) at the active site of arginase 1 enhances stability and catalytic activity at physiological pH. PEGylation also extends the circulation half-life (t 1 / 2) of recombinant arginase 1.

[0011] In various embodiments, the methods comprise 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). The Co-arginase 1 intermediate can be purified by a purification process comprising one or more of the following steps: cell disruption by high-pressure homogenization, homogenate cleanup, SP Sepharose FF cation exchange capture chromatography, cobalt exchange, ultrafiltration / diafiltration, Q Sepharose FF anion exchange flow-through chromatography, Capto MMC multi-layer chromatography, and ultrafiltration / diafiltration. The purified Co-arginase 1 intermediate can be processed to form a PEGylated drug substance or frozen and stored for subsequent conversion into a drug substance.

[0012] In preferred embodiments of the methods, E. coli lysate containing rhARG1 is loaded onto a cation exchange (CEX) chromatography column (also referred to as "column 1") to capture rhARG1, and then eluted using a high salt solution to provide a first protein product ("first protein product").

[0013] In one or more embodiments, the method further comprises loading the first protein product onto an anion exchange (AEX) chromatography column (also referred to as "column 2") and collecting the flow-through to provide a second protein product ("second protein product"). In another aspect of these methods, the method further comprises loading the second protein product onto a multicomponent chromatography (MMC) column, which captures arginase 1 and then elutes to provide a third protein product ("third protein product"). In certain embodiments, this third chromatography column (also referred to as "column 3") may be a size exclusion chromatography (SEC) column.

[0014] Various embodiments involve replacing the natural manganese coenzyme of argininase with a cobalt coenzyme. Cobalt substitution (also known as cobalt loading) can be performed at any step in the manufacturing process. For example, cobalt loading of argininase 1 can be performed on an E. coli lysate, a first protein product, a second protein product, a third protein product, or at any step associated with PEGylated argininase 1. In other embodiments, cobalt loading can be performed on argininase 1 eluted from column 1, argininase 1 eluted from column 2, or argininase 1 eluted from column 3. Cobalt loading can also be performed on argininase 1 eluted from a CEX column, an AEX column, an MMC column, or an SEC column.

[0015] Arginase 1 can be cobalt-loaded using a variety of cobalt-containing solutions and at various temperatures. In one or more embodiments, the cobalt salt comprises Co2+, such as CoCl2. In a preferred embodiment, arginase 1 is cobalt-loaded using CoCl2 at or about room temperature (e.g., from about 15°C to about 25°C or from about 20°C to about 25°C). The rate of cobalt loading can be controlled by increasing or decreasing the reaction temperature. Cobalt loading can also be performed at a range of pH values.

[0016] One aspect of the present disclosure involves modifying the conditions associated with CEX chromatography (column 1). The amount of protein loaded onto column 1 can be increased or decreased to select for different arginase 1 charge variants. The loading factor can be controlled to favor a more desirable CEX charge species profile. A loading factor (amount of protein (grams) / volume of CEX column resin (liters)) of up to about 60 g / L can produce arginase 1 with high specific activity. In various embodiments, the loading factor is 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.

[0017] In a preferred embodiment, arginase 1 is first captured on column 1, followed by purification on columns 2 and 3. In an alternative embodiment, E. coli lysate can be loaded onto an AEX column (e.g., column 2), and the flow-through applied to a CEX column to capture arginase 1. In another embodiment, cobalt loading of arginase 1 can occur after the PEGylation reaction. Alternatively, other chromatography columns, such as SEC columns, can be used instead of MMC columns.

[0018] Human arginase 1 and 2 proteins subjected to the methods of the present invention have two Mn 2+ sites; either or both sites can be substituted to generate modified arginase 1 and 2 proteins with non-native metal cofactors. In certain embodiments, the protein exhibits a k cat / KM greater than 200 mM s 1 at pH 7.4. In a specific embodiment, the protein exhibits a k cat / KM in the range of about 200 mM s 1 to about 4,000 mM s 1 at pH 7.4. In another embodiment, the protein exhibits a k cat / KM in the range of about 400 mM s 1 to about 2,500 mM s 1 at pH 7.4 and 37°C. In a specific embodiment, the present invention contemplates a protein comprising the amino acid sequence of human arginase 1 or 2 and a non-natural metal cofactor, wherein the protein exhibits a kcat / KM greater than 400 mM-1s-1 at 37° C. and pH 7.4. Exemplary kcat / KM values at pH 7.4 and 37° C. include about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 800, about 900, about 1,000, about 1,100, about 1,200, about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, and about 4,000 mM-1s-1.

[0019] [Recombinant human arginase] [1] [, Pharmaceutical Compositions and Formulations]

[0020] Another aspect of the present invention relates to rhARG1, Co-rhARG1 and / or Co-rhARG1-PEG produced by the methods described herein.

[0021] Another aspect of the present invention relates to pharmaceutical compositions comprising rhARG1, Co-rhARG1, and / or Co-rhARG1-PEG and a pharmaceutically acceptable 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 approximately 50 mM NaCl, approximately 1 mM K2HPO4, approximately 4 mM KH2PO4, and approximately 1.5% w / v glycerol.

[0022] [Recombinant human arginase] [1] [Administration]

[0023] Another aspect of the present invention relates to the administration of recombinant human arginase 1 (e.g., Co-rhARG1-PEG). Administration can be by any suitable method, including intravenous or subcutaneous administration. In one or more embodiments of this aspect, the dosage of Co-rhARG1-PEG is determined by a specific algorithm:

[0024] In one or more embodiments of this algorithm, patients begin therapy at 0.10 mg / kg. Monitor plasma arginine levels. If plasma arginine levels are >150 μM, increase the dose to 0.20 mg / kg. If plasma arginine levels are <50 μM, reduce the dose to 0.05 mg / kg. Otherwise, maintain the patient at 0.10 mg / kg.

[0025] In one or more embodiments of this algorithm, the dose modification is as follows: ● If the plasma arginine level is >150 μM, then if the 2 doses prior to a single 168-hour sample were a) the same dose level expressed in mg / kg and b) consecutive (no missed doses), this sample will be used to increase the dose by 2 dose levels as shown in the table below (not to exceed 0.20 mg / kg). • If plasma arginine levels from 2 consecutive 168-hour samples (regardless of whether a dose was missed) are < 50 μM, reduce the dose by 1 dose level from the table below, but not to below 0.05 mg / kg. [Dose Level] [ , (a) , ] [dose] 1 (minimum possible dose) 0.05 mg / kg 2 (starting dose) 0.10 mg / kg 3 0.15 mg / kg 4 (maximum possible dose) 0.20 mg / kg a) Pegylated arginine should be administered starting at Level 2 (0.10 mg / kg). If necessary, the dose may be increased by 2 dose levels. The dose may be decreased by 1 dose level.

Brief description of the diagram

[0026] Other features of the present invention will become apparent from the following written description and accompanying drawings, in which: Figure 1 shows the amino acid and DNA sequences of arginase 1 and the amino acid sequence of arginase 2. Figure 1(a) shows the amino acid sequence of recombinant human arginase 1 expressed in Escherichia 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 native human arginase 1 monomer. Figure 1(c) shows the amino acid sequence of arginase 2 lacking the N-terminal methionine found in the native human arginase 2 monomer. FIG. 2 is a schematic diagram of an exemplary process for fermenting E. coli and expressing arginase 1. Figure 3 is a schematic diagram of an exemplary process for purifying recombinant human arginase 1, cobalt-substituted recombinant human arginase 1, and PEGylated cobalt-substituted recombinant human arginase 1. Figure 3(a) illustrates an exemplary process including a cation exchange column (column 1), an anion exchange column (column 2), and a Capto multi-column column (column 3), as well as a cobalt loading step. Figure 3(b) illustrates PEGylation of the Co-arginase 1 intermediate, followed by final filtration and formulation to provide the drug substance. Figure 4 shows the column chromatography purification of arginase 1. Figure 4(a) shows that E. coli cell lysate was loaded onto a cation exchange column (column 1), washed, and then eluted with a high salt solution (to provide the first protein product). Protein loading and elution were assessed by measuring UV absorbance at 280 nm. Approximately 3 liters (L) of cell lysate was 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 that arginase 1 (the first protein product) eluted from column 1 was loaded onto an anion exchange column (column 2), and protein concentration was measured using absorbance at 280 nm. Arginase 1 was collected in the flow-through from column 2 to provide the second protein product. FIG4( c ) shows that arginase 1 was captured on a Capto polycation exchange column (column 3) and eluted using a high salt solution to provide a third protein product. Figure 5 shows the results of an analytical cation-exchange HPLC method used to determine the charge heterogeneity profile of a sample of the Co-arginase 1 intermediate (also referred to as the first protein product) eluted from column 1. A 1 mg / mL sample of arginase 1 was loaded onto a cation-exchange column with a mobile phase of 20 mM MES, pH 6.0 buffer at a flow rate of 1.0 mL / min. A gradient of 0-500 mM NaCl was introduced over 40 minutes, and the amount of protein eluted from the column was estimated by absorbance at 280 nm. Figure 5(a) shows a representative chromatogram of charge heterogeneous species of arginase 1. Arginase 1 charge variants eluted from the analytical HPLC column after 10-20 minutes. Figure 5(b) shows the same chromatogram as Figure 5(a), except for a greater magnification of the peaks. Figure 5(c) shows the peak assignments for the cation exchange charge variant of arginase 1. Figure 5(d) shows the typical charge heterogeneity profile of a drug substance resolved by imaging capillary isoelectric focusing (iCIEF). Figure 6 shows the results of an LC / MS method used to characterize glucuronidation variants of arginase 1 produced by expressing rhARG in Escherichia coli. LC / MS analysis identified unmodified arginase 1 (monomer), glucuronidated arginase 1, phosphogluconidated arginase 1, and 2X glucuronidated arginase 1. Traces are from two separate production experiments of the drug intermediate. Mass spectra were overlaid within 33-35 minutes on a RP LCMS at 35°C; spectra were normalized to the peak intensity of the signal produced by unmodified arginase 1. The peak intensity of each variant is proportional to the relative abundance. Figure 7 shows the results of applying a 0.0-0.2 M NaCl gradient to Column 1. Fractions were collected every 0.25 CV (column volume) throughout the gradient. The data represent two experiments with Column 1 using two different batches of harvested cell slurry as feed. The load factor used for evaluation was 30 g / L. This gradient successfully separated the various gluconated species while maintaining product recovery. Figure 8 shows the enzymatic activities of the Co-argininase 1 intermediate and the Co-rhARG1-PEG drug substance. Figure 8(a) shows a representative enzyme kinetic analysis of the Co-argininase 1 intermediate (conversion of arginine to ornithine at 37°C with substrate concentrations ranging from 0 to 2 mM). Figure 8(b) shows a representative enzyme kinetic analysis of the Co-rhARG1-PEG drug substance. Figure 9 shows a pharmacokinetic analysis of the Co-rhARG1-PEG drug substance. Figures 9(a) and (b) show the mean (±SD) arginase 1 concentration versus time profiles in patients after administration of a single intravenous dose of Co-rhARG1-PEG: Part 1. Linear (a) and semi-logarithmic (b) plots are shown. Note that the first mean BQL concentration is plotted at half the LLOQ (0.125 µg / mL). Mean circulating drug concentrations in all patients increased with increasing Co-rhARG1-PEG dose. Figures 9(c)-9(f) show the mean (±SD) Co-rhARG1-PEG concentration versus time profiles in patients after administration of weekly (weekly) intravenous doses of Co-rhARG1-PEG: Part 2. Linear plots of week 1 (c) and week 8 (d) and semi-logarithmic plots of week 1 (e) and week 8 (f) are shown. Figures 10(a) through 10(c) show three representative composite plots of pharmacokinetics (PK), pharmacodynamics (PD), and anti-drug antibodies (ADA) from a Phase 1 / 2, open-label study evaluating Co-rhARG1-PEG administration in patients with arginase 1 deficiency. Both Co-rhARG1-PEG and anti-PEG titers were measured. Using escalation stopping criteria, the doses set 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 Part 2 period shown). Applying the dose-escalation stopping criteria allowed the remaining patients in the trial to be set at the various dose levels used in Part 2. These same criteria can be used to adjust (increase or decrease) the dose of any patient already taking Co-rhARG1-PEG in response to arginine levels shifting outside the optimal (healthy) range. Figure 11 shows a comparison of intravenous and subcutaneous administration of Co-rhARG1-PEG. Optimal plasma arginine concentrations in patients range from 40 μM to 115 μM (dashed line). Subcutaneous administration of Co-rhARG1-PEG maintains arginine concentrations within this optimal range for a longer period compared to intravenous administration. Figure 11(a) includes data from the first week after the completion of Part 2, and Figure 11(b) excludes this extended intravenous data for the first week. The graph shows the mean values across patients, and the data were obtained based on the dose determined by each patient's stopping criteria. Figure 12 shows plasma arginine and plasma guanidino compound levels after administration of Co-rhARG1-PEG. Figure 12(a) shows plasma arginine levels at baseline, after dose 1, after dose 8, and during the open-label extension (OLE). Figure 12(b) shows plasma levels of guanidinoacetic acid (GAA), N-α-acetyl-L-arginine (NAA), α-keto-δ-guanidinovaleric acid (GVA), and arginine (ARGA) at baseline and during the OLE. Figure 13 shows baseline deficits and responses to clinical outcomes. Figure 13(a) shows baseline deficits in patients with arginase 1 deficiency on the 6-Minute Walk Test (6MWT), Gross Motor Function Measure (GMFM) sections D and E, and the Adaptive Behavior Assessment System (ABAS). Figure 13(b) shows clinical responses on the 6MWT, GMFM-D, and GMFM-E. Figure 14 shows time-dependent improvements in the 6MWT, GMFM-D, and GMFM-E. Figure 14(a) shows the percentage of clinical responders to the 6MWT among all patients and those with baseline deficits at doses 8 and 20. Figure 14(b) shows the percentage of clinical responders to GMFM-D among all patients and those with baseline deficits at doses 8 and 20. Figure 14(c) shows the percentage of clinical responders to GMFM-E among all patients and those with baseline deficits at doses 8 and 20. FIG15 shows site-specific PEGylation analysis of three batches of Co-rhARG1-PEG. Implementation Method

[0027] [Recombinant human arginase] [1]

[0028] Human arginase 1 (designated hArg1) is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine (L-Arg) to produce L-ornithine and urea. Argininase 1 is a trimer of three non-covalently bound identical monomeric units. Monomeric argininase 1 is enzymatically active but relatively unstable. Substituting cobalt (Co 2+ ) for native manganese (Mn 2+ ) in the active site of argininase 1 enhances catalytic activity at physiological pH. The methods described herein for producing cobalt-substituted argininase 1 provide highly pure and active enzymes. These methods also provide Co-argininase 1 (Co-rhARG1) as an isolated intermediate in the manufacture of drug substances. In one or more embodiments, the drug substance is PEGylated Co-argininase 1 (Co-rhARG1-PEG). PEGylation of Co-argininase 1 significantly extends its circulation half-life. Likewise, although specific reference is made herein to rhARG1, the methods, formulations, and uses described herein are also applicable to rhARG2.

[0029] As used herein, the term "rhARG1" refers to recombinant human arginase 1, eg, a recombinant enzyme having at least 98% sequence identity to SEQ ID NO: 1.

[0030] As used herein, the terms "Co-rhARG1," "Co-arginase 1 intermediate," and the like refer to rhARG1 in which at least some of the native manganese cofactor is replaced by cobalt. In one or more embodiments, Co-rhARG1 is an isolatable intermediate in the production and / or purification of Co-rhARG1-PEG.

[0031] As used herein, the terms "Co-rhARG1-PEG," "PEGylated Co-argininase 1," and the like refer to Co-rhARG1 having one or more PEG units covalently attached to the enzyme, e.g., at the N-terminal amino acid and / or the free amines of one or more lysine residues.

[0032] The amount of Co-rhARG1-PEG drug substance can be expressed as the mass of unPEGylated enzyme. In one embodiment of this method, each mg (based on enzyme) of Co-rhARG1-PEG drug substance also contains approximately 1-2 mg of PEG (e.g., approximately 1.4 mg of PEG).

[0033] 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). Overlapping oligonucleotides were used in a PCR reaction to generate codon-optimized arginase 1 DNA 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 native human arginase 1 monomers. The calculated molecular weight of co-arginase 1 is 34,721.6 daltons (Table 1). The calculated molecular weight of the homotrimeric co-arginase 1 is 104,164.8 daltons. Arginase 1 does not possess any disulfide bonds.

[0034] Table 1: Structural information of exemplary Co-arginase 1 intermediates Molecular weight of Co-arginine kinase 1 34721.6 Daltons (calculated value with cobalt) Homotrimeric Co-argininase 1 104164.8 Daltons (calculated value with cobalt) Monomer length 321 amino acid residues disulfide bonds none Amino acid sequence The primary sequence of recombinant human arginase 1 is provided in Figure 1 .

[0035] In one or more embodiments, the calculated molecular weight of monomeric Co-rhARG1-PEG is about 75-115 kDa. In one or more embodiments, the calculated molecular weight of 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 per mole of Co-arginase 1 monomer, for example, about 8 to about 16 moles of PEG per 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 per 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, for example, 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 specific embodiment, the average MW of the PEG is about 5,000 Daltons.

[0036] In one or more embodiments, Co-rhARG1-PEG comprises PEGylated arginine. PEGylated arginine has the following two chemical names: a. Poly(oxy-1,2-ethanediyl), α-(carboxymethyl)-ω-methoxy-, amido-arginine kinase 1 [cobalt cofactor] (synthetic human) (1:10), trimer b. Des-Met 1-argininase-1 (active argininase, EC 3.5.3.1) (Homo sapiens), in which manganese is replaced by cobalt, and an average of 10 primary amines (the N-terminal serine and one of the N-6-lysine primary amines) are acetylated with [methoxypoly(ethyleneoxy)]acetyl. This non-covalent homotrimer is produced in Escherichia coli. The molecular formula of polyethylene glycol argininase is C₁₅H₂₄N₁₁₆O₄₅S₆[C₃H₄O₂(C₂H₄O)₄]₂a monomer. The average molecular weight of the polyethylene glycol argininase trimer is 284 kDa. The CAS registry number for polyethylene glycol argininase is 1659310-95-8.

[0037] Potential PEGylation sites for PEGylated arginine are shown below:

[0038] 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 certain 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 certain embodiments, Co-rhARG1-PEG is pegylated at the amino acid residues K222 and / or K223. In certain embodiments, Co-rhARG1-PEG is not PEGylated at K222 and / or K223 amino acid residues. In certain embodiments, Co-rhARG1-PEG is not PEGylated at one or more of K3, K149, K190, K195, K29, K265, and K283 amino acid residues. In certain embodiments, Co-rhARG1-PEG is not PEGylated at K3, K149, K190, K195, K29, K265, and K283 amino acid residues.

[0039] In one or more embodiments of Co-rhARG1-PEG, K16 is PEGylated in a range of about 15% to about 60%. In one or more embodiments of Co-rhARG1-PEG, K32 is PEGylated in a range of about 35% to about 80%. In one or more embodiments of Co-rhARG1-PEG, K38 is PEGylated in a range of about 20% to about 85%. In one or more embodiments of Co-rhARG1-PEG, K40 is PEGylated in a range of about 10% to about 60%. In one or more embodiments of Co-rhARG1-PEG, K47 is PEGylated in a range of about 10% to about 60%. In one or more embodiments of Co-rhARG1-PEG, K67 is PEGylated in a range of about 40% to about 90%. In one or more embodiments of Co-rhARG1-PEG, K74 is PEGylated at a range of about 30% to about 95%. In one or more embodiments of Co-rhARG1-PEG, K82 is PEGylated at a range of about 30% to about 98%. In one or more embodiments of Co-rhARG1-PEG, K87 is PEGylated at a range of about 15% to about 65%. In one or more embodiments of Co-rhARG1-PEG, K88 is PEGylated at a range of about 25% to about 70%. In one or more embodiments of Co-rhARG1-PEG, K152 is PEGylated at a range of about 25% to about 85%. In one or more embodiments of Co-rhARG1-PEG, K154 is PEGylated at a range of about 15% to about 65%. In one or more embodiments of Co-rhARG1-PEG, K171 is PEGylated within a range of about 20% to about 75%. In one or more embodiments of Co-rhARG1-PEG, K222 is PEGylated within a range of 0% to about 30%. In one or more embodiments of Co-rhARG1-PEG, K223 is PEGylated within a range of 0% to about 35%. In one or more embodiments of Co-rhARG1-PEG, K312 is PEGylated within a range of 0% to about 45%. In one or more embodiments of Co-rhARG1-PEG, K321 is PEGylated within a range of 0% to about 45%.

[0040] The PEG-to-protein molar ratio is a property that indicates the degree of PEGylation. In one or more embodiments, about 1 mole to about 20 moles of PEG PEGylates one mole of Co-rhARG1. Exemplary ranges of PEG:Co-rhARG1 molar ratios 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 certain embodiments, the PEG:Co-rhARG molar ratio ranges from about 7 mol / mole to about 15 mol / mole.

[0041] Free PEG was measured to demonstrate PEG clearance and stability. In certain embodiments, the free PEG concentration (µg) in PEGylated Co-rhARG1 (mL) is less than or equal to 500 µg / mL, less than or equal to 400 µg / mL, less than or equal to 300 µg / mL, less than or equal to 200 µg / mL, less than or equal to 100 µg / mL, and less than or equal to 50 µg / mL.

[0042] Human arginase 1 catalyzes the fifth and final step in the urea cycle, the conversion of L-arginine to L-ornithine and urea. The PEGylated drug substance, Co-rhARG1-PEG, catalyzes the same reaction. The assay used to assess enzyme activity measures the conversion of L-arginine to L-ornithine at pH 7.4 and 37°C over a fixed reaction time. Product conversion is converted to a reaction rate and fitted to the Michaelis-Menten equation to determine Km and kcat.

[0043] V max is the maximum reaction rate achieved at saturating substrate concentration; K m is the Michaelis-Menten binding constant, which measures the substrate concentration that produces a rate half that of V max. The enzymatic turnover rate, k cat, is calculated as V max / [E].

[0044] Specific activity was determined by dividing the reaction velocity (expressed in μmol / min) at 2 mM arginine by the enzyme concentration (expressed in mg).

[0045] The KM and kcat values for the Co-rhARG1-PEG drug substance typically ranged from 0.15 to 0.22 mM and approximately 200 to 300 / sec, respectively, as measured in enzyme activity assays. Following PEGylation of the Co-arginase 1 intermediate to form the drug substance, enzymatic activity was not significantly altered compared to the unPEGylated intermediate. However, PEGylation significantly increased the circulation half-life of the Co-rhARG1-PEG drug product compared to the Co-arginase 1 intermediate.

[0046] In one or more embodiments, the protein (e.g., Co-rhARG1 or Co-rhARG1-PEG) exhibits a kcat / KM greater than 200 mM s at pH 7.4. In a specific embodiment, the protein exhibits a kcat / KM in the range of about 200 mM s to about 4,000 mM s at pH 7.4. In another embodiment, the protein exhibits a kcat / KM in the range of about 400 mM s to about 2,500 mM s at pH 7.4 and 37°C. In a specific embodiment, the present invention contemplates a protein comprising the amino acid sequence of human arginase 1 and a non-natural metal cofactor, wherein the protein exhibits a kcat / KM greater than 400 mM s at 37°C and pH 7.4. Exemplary kcat / KM values at pH 7.4 and 37°C include about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 800, about 900, about 1,000, about 1,100, about 1,200, about 1,500, about 2,000, about 2,500, about 3,000, about 3,500, and about 4,000 mM-1s-1, or any range therebetween.

[0047] Specific activity indicates the efficacy of a protein (eg, 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.

[0048] In one or more embodiments, rhARG1, Co-rhARG1, or Co-rhARG1-PEG may have at least 98%, 98.5%, 99%, or 99.5% identity to 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 set forth in SEQ ID NO: 1. Various alignment algorithms and / or programs can be used to calculate the identity between two sequences, including FASTA or BLAST, available at the National Center for Biotechnology Information website (http: / / www.ncbi.nlm.nih.gov / ).

[0049] In one or more embodiments, rhARG1, Co-rhARG1, or Co-rhARG1-PEG comprises at least one amino acid substitution at a position selected from H100, D123, H125, D127, D231, D233, D180, S229, and C302. In certain 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.

[0050] Using the methods described herein, cobalt can be used to replace substantially all of the manganese cofactor in arginase 1. The substitution of a cobalt cofactor shifts the K m of arginine from 2.8 mM to approximately 0.18 mM at pH 7.4. In one or more embodiments, Co-rhARG1-PEG comprises from about 0.1 μg to about 2 μg Co / mg protein. Exemplary cobalt loadings include about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about 1.9, and about 2 μg Co / mg protein.

[0051] Free cobalt was measured to demonstrate cobalt clearance and stability. In certain 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.

[0052] The total amount of cobalt affects protein efficacy and is indicative of bound cobalt, as the amount of free cobalt is relatively small. In certain embodiments, the total cobalt concentration is in the range of about 5 μg / mL to about 20 μg / mL, about 6 μg / mL to about 20 μg / mL, about 7 μg / mL to about 20 μg / mL, about 8 μg / mL to about 20 μg / mL, about 9 μg / mL to about 20 μg / mL, about 5 μg / mL to about 19 μg / mL, about 6 μg / mL to about 19 μg / mL, about 7 μg / mL to about 19 μg / mL, about 8 μg / mL to about 19 μg / mL, about 9 μg / mL to about 19 μg / mL, about 5 μg / mL to about 18 μg / mL, about 6 μg / mL to about 18 μg / mL, about 7 μg / mL to about 18 μg / mL, about 8 μg / mL to about 18 μg / mL, about 9 μg / mL to about 18 μg / mL, about 5 μg / mL to about 17 μg / mL. µg / mL, about 6 µg / mL to about 17 µg / mL, about 7 µg / mL to about 17 µg / mL, about 8 µg / mL to about 17 µg / mL, about 9 µg / mL to about 17 µg / mL, about 5 µg / mL to about 16 µg / mL, about 6 µg / mL to about 16 µg / mL, about 7 µg / mL to about 16 µg / mL, about 8 µg / mL to about 16 µg / mL, about 9 µg / mL to about 16 µg / mL, about 5 µg / mL to about 15 µg / mL, about 6 µg / mL to about 15 µg / mL, about 7 µg / mL to about 15 µg / mL, about 8 µg / mL to about 15 µg / mL, and about 9 µg / mL to about 15 µg / mL.

[0053] In various embodiments, the Co-rhARG1-PEG comprises less than about 1 μg Mn / mg protein, for example, 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 drug substance contains about 2 μg Co / mg protein and about 0.05 μg Mn / mg protein.

[0054] In various embodiments, Co-rhARG1-PEG comprises less than about 1 μg Fe / mg protein, for example, 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.

[0055] [rhARG1] [、] [Co-rhARG1] [and] [PEGrhARG1] [Production and purification]

[0056] Overviews of exemplary upstream and downstream production methods can be seen in Figures 2 and 3.

[0057] [Shake flask expansion]

[0058] The purpose of shake flask expansion / fermentation is to generate inoculum for inoculating production fermenters. Shake flask expansion produces cell pellets for inoculating production reactors and also for analytical purposes. A representative overview of the Arginase 1 fermentation process can be seen in Figure 2.

[0059] Aliquots of the inoculum medium were introduced into one 500 mL flask (primary flask) and six 3 L disposable flasks (secondary flasks). The flasks were autoclaved and the sterile supplement was transferred to each flask. Prior to inoculation, the primary culture medium was preheated to a processing temperature of 37°C. Prior to secondary inoculation, the secondary flasks were preheated to a processing temperature of 37°C.

[0060] Remove a vial of Arginase 1, representing an E. coli working cell bank (WCB), from the refrigerator and thaw. Aseptically add the target volume of thawed cells (approximately 1.1 mL) to the primary flask and incubate the flask at 37°C with agitation. Beginning several hours after inoculation, remove samples from the flask hourly to track cell growth by optical density at 600 nm (OD600). Once a target OD600 of ≥ 1.0 is reached in the primary flask, aseptically transfer the target volume (15 mL) of the primary culture to each secondary flask. Incubate the secondary flasks at 37°C with agitation. Beginning 4 hours after inoculation, remove samples from one secondary flask hourly, increasing this rate to every 30 minutes after the OD600 reaches ≥ 1.5. Sample the remaining secondary flasks when the measurement meets the designated density of OD600 ≥ 2.0. If the average OD600 of all secondary flasks meets the specified transfer criteria, the flasks are pooled and the inoculum is transferred to the production fermentor. A representative overview of the fermentation process for arginase 1 can be seen in Figure 2.

[0061] [Producing fermentation]

[0062] The purpose of the production fermentation is to expand the shake flask culture and induce arginase 1 production. Production fermentation can produce large amounts of arginase 1. After a shake flask expansion phase to establish a cell mass, the fermentation process produces arginase 1 (in E. coli) as a soluble protein. In one embodiment, a 1500 L fermentor contains a primary batch culture medium including sterile pre-inoculation additives. After inoculation, fermentor inputs include nutrient feed, defoaming solution, and acid or base added to maintain culture pH. A secondary vessel holds the nutrient feed medium. An automated control strategy maintains key parameters for continuous cell growth, including dissolved oxygen, aeration rate, agitation rate, pH, pressure, and temperature. Arginase 1 expression is induced by adding IPTG (isopropyl β-D-1-thiogalactopyranoside), and harvesting is performed approximately 18 hours later. At the end of production, fermentor performance is evaluated by monitoring cell density, percent solids, and the proportion of soluble arginase 1.

[0063] In a preferred embodiment, the fermentation medium is prepared directly in the production fermentor. Purified water is added to the fermentation medium to the desired weight prior to sterilization-in-place (SIP). Once the medium has cooled, the sterile additions of kanamycin, glucose, and potassium phosphate are immediately filter-sterilized and added to the production fermentor. If necessary, the sterile medium is filtered through a 0.2 µm sterile filter and brought to the desired pre-inoculation weight with purified water. The fermentation medium is titrated to a controlled pH using base (ammonium hydroxide).

[0064] The pooled inoculum was aseptically inoculated into a production fermenter at 37°C via pressure-assisted transfer. From the time of inoculation until the fermentation broth cooled, fermentation broth samples were collected regularly and measured for OD600 analysis. Glucose samples were obtained at regular intervals starting 3 hours after inoculation and increased in frequency after 9 hours. Antifoaming solution was added as needed during the fermentation process to prevent excessive foaming of the culture. Dissolved oxygen was controlled by high-pressure agitation and aerated as needed. Acid and base inputs were used to maintain the culture pH. The growth medium was preferably maintained at 36-38°C and a pH of 7.0-7.4 with agitation and aeration.

[0065] The nutrient feed consists of yeast extract, Martone B-1, L-cysteine hydrochloride, and glycerol. Feeding begins when the glucose concentration is less than 10 g / L (12-14 hours after inoculation) and continues at a constant rate until the production period is complete. Expression is triggered by the addition of IPTG. Induction is continued for 18 hours. After the fermentation is complete, the fermentation is cooled and prepared for harvesting. The production fermentor produces approximately 6 g / L of soluble arginase 1. An overview of the production fermentation can be seen in Figure 3.

[0066] [Harvest Operation]

[0067] Harvesting procedures can capture cells containing soluble arginase 1, disrupt / lyse the cells, and remove the lysate from cell debris using centrifugation and / or filtration. The recovered cell slurry can be frozen or kept at low temperatures for long-term storage. Harvesting procedures can involve collecting cells by centrifugation, lysing them twice using a homogenizer or disrupting them under pressure (French press), centrifuging them a second time, and then filtering them through a membrane prior to the first chromatography step.

[0068] 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 (pH 7.6) and then passed through a homogenizer twice. The pH of 25 mM HEPES can also be between pH 7.2 and 7.6. The lysed material is clarified using a centrifuge to remove cell debris and then membrane filtered through a 0.2 μm filter. In a preferred embodiment, the harvesting step is performed at a target temperature of ≤ 15°C.

[0069] In an alternative embodiment, cell disruption is performed using high pressure. The cell slurry is transferred to a homogenizer at a controlled rate, and the homogenization effluent is passed through a heat exchanger to reduce the temperature increase seen during pressure homogenization. Frozen cells are homogenized twice. The first lysis pool is transferred from the collection vessel back to the feed vessel. The holding time between each pass is minimized to reduce potential microbial growth.

[0070] The lysed material is purified by centrifugation to remove cell debris from the soluble lysate fraction. The lysate is transferred to a disc-type, intermittent exhaust centrifuge at a controlled rate. The purified lysate is collected for further processing.

[0071] Filter the purified lysate using, for example, a 0.2 µm filter. During process operation, a process change filter can also be used to control microorganisms. For this purpose, the filter can be a 0.5 µm or 0.2 µm filter. This step also removes small particles from the purified material that could not be separated during the purification operation. Before use, rinse the filter thoroughly with purified water and equilibrate it with 25 mM HEPES (pH 7.6) buffer. Prefiltration can be performed before each downstream process step to reduce potential bioburden.

[0072] [rhARG1] [、] [Co-rhARG1] [and] [Co-rhARG1-PEG] [Purification]

[0073] 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 cobalt loading to produce Co-rhARG1 and / or reaction with a PEGylation reagent to provide Co-rhARG1-PEG.

[0074] Various embodiments of the purification process involve the use of a cation exchange (CEX) column to capture rhARG1. In one or more embodiments, the CEX column is the first column ("column 1") used in combination with multiple chromatography columns. The protein product eluted from column 1 is the "first protein product."

[0075] In one or more embodiments, column 1 uses cation exchange chromatography to bind rhARG1 at a pH ranging from about 7 to about 8 (e.g., about pH 7.6). In one or more embodiments, rhARG1 is bound in the absence of salt or at low salt concentrations. In one or more embodiments, rhARG1 is eluted using a buffer having a high salt (e.g., NaCl) concentration (e.g., 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.

[0076] In various embodiments, a salt gradient is used to separate the different charge variants of rhARG1. Exemplary salt gradients are from about 0 M to about 0.5 M NaCl, from about 0 M to about 0.4 M NaCl, from about 0 M to about 0.3 M NaCl, from about 0 M to about 0.2 M NaCl, or from about 0 M to about 0.1 M NaCl.

[0077] In one or more embodiments, the method further comprises loading the first protein product (optionally after cobalt substitution) onto an anion exchange (AEX) chromatography column ("column 2") and collecting the flow-through to provide a second protein product ("second protein product"). In another aspect of these methods, the method further comprises loading the second protein product onto a third column, which captures arginase 1 and is then eluted to provide a third protein product ("third protein product"). In certain embodiments, this third chromatography column ("column 3") can be a size exclusion chromatography (SEC) column or a multicomponent chromatography (MMC) column.

[0078] In various embodiments, rhARG1 is loaded with Co to replace the Mn cofactor. In one or more embodiments, Co loading is performed using a Co2+ salt (e.g., CoCl2). Incubation time is temperature-dependent, such that lower cobalt substitution temperatures require longer incubation times and higher cobalt substitution temperatures do not require longer incubation times. Cobalt loading temperatures can be as low as 1°C or greater than 50°C, and corresponding incubation times can be as long as 8 hours or longer or less than 10 minutes.

[0079] In various embodiments, rhARG1 or Co-rhARG1 is reacted with a PEGylation reagent, such as methoxy PEG succinimidyl carboxymethyl ester (MW 5000). The PEGylation reagent is typically provided at a 10-40 molar excess relative to the enzyme. The incubation time can range from 0.5 to 4 hours. The pH during PEGylation can be between about 8 and about 9, for example, about pH 8.4.

[0080] In one or more embodiments, the purified PEGylated protein rhARG1 or Co-rhARG1 comprises arginase I monomer, gluconylated arginine I, phosphogluconylated arginine I, 2× gluconylated arginine I, gluconylated + phosphogluconylated arginine I, and 2× phosphogluconylated arginine I.

[0081] In certain embodiments, the purified PEGylated rhARG1 or Co-rhARG1 protein comprises at least 70% arginase I monomers. Exemplary amounts include at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% arginase I monomers. In certain embodiments, the purified PEGylated rhARG1 or Co-rhARG1 protein comprises less than 10% gluconylated arginase I. Exemplary amounts include about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, or about 9% gluconylated arginase I, or any range therebetween. In certain embodiments, the purified PEGylated rhARG1 or Co-rhARG1 protein comprises less than 10% phosphogluconylated arginase I. Exemplary amounts include about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, or about 9% phosphogluconylating arginine I. In certain embodiments, the purified PEGylated protein rhARG1 or Co-rhARG1 comprises at least 70% arginine I monomer, less than 10% gluconylating arginine I, and less than 10% phosphogluconylating arginine I.

[0082] Imaged capillary isoelectric focusing (iCIEF) can measure the identity of PEGylated proteins (due to the degree of heterogeneity within the PEGylated trimer). In one or more embodiments, iCIEF analysis of purified PEGylated rhARG1 or Co-rhARG1 protein contains nine distinct peaks: Peak 1, Peak 2, Peak 3, Peak 4, Peak 5, Peak 6, Peak 7, Peak 8, and Peak 9. Each peak corresponds to nine differently charged species: Species 1, Species 2, Species 3, Species 4, Species 5, Species 6, Species 7, Species 8, and Species 9. The area under the curve for each peak corresponds to the proportion of that specific species. In one or more embodiments, certain peaks of related species may be grouped together, for example, Peaks 1+2 or Peaks 3+4.

[0083] In certain embodiments, the purified PEGylated protein rhARG1 or Co-rhARG1 comprises an area under the curve of Peak 1 of less than about 30%, less than about 25%, less than about 20%, less than about 15%, and less than about 10%.

[0084] In certain embodiments, the purified PEGylated protein rhARG1 or Co-rhARG1 comprises an area under the curve of Peak 2 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%.

[0085] In certain embodiments, the purified PEGylated protein rhARG1 or Co-rhARG1 comprises a combined peak 1+2 area under the curve 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%.

[0086] In certain embodiments, the purified PEGylated protein rhARG1 or Co-rhARG1 comprises an area under the curve for Peak 3+4 within the following ratios: 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%.

[0087] In certain embodiments, the purified PEGylated protein rhARG1 or Co-rhARG1 comprises an area under the curve for Peak 5 within the following ratio ranges: 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%, 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%.

[0088] In certain embodiments, the purified PEGylated protein rhARG1 or Co-rhARG1 comprises an area under the curve for Peak 6 within the following ratios: 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%.

[0089] In certain embodiments, the purified PEGylated protein rhARG1 or Co-rhARG1 comprises an area under the curve of Peak 7 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%.

[0090] In certain embodiments, the purified PEGylated protein rhARG1 or Co-rhARG1 comprises an area under the curve of Peak B of less than about 25%, less than about 20%, less than about 15%, less than about 10%, and less than about 5%.

[0091] In certain embodiments, the purified PEGylated protein rhARG1 or Co-rhARG1 comprises an area under the curve of Peak 9 of 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%.

[0092] [rhARG1] [、] [Co-rhARG1] [and] [Co-rhARG1-PEG] [Administration]

[0093] As described herein, rhARG1, Co-rhARG1, and Co-rhARG1-PEG (and compositions comprising the same) can 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 (and compositions comprising the same) are administered intravenously (IV) or subcutaneously (SC).

[0094] Compositions containing rhARG1, Co-rhARG1, and Co-rhARG1-PEG can be provided in the form of formulations with physiologically tolerable liquid, gel, or solid carriers, diluents, and excipients. These compositions are typically prepared as liquid solutions or suspensions (e.g., injectable solutions). Suitable diluents and excipients are, for example, water, saline, dextrose, glycerol, or the like, and combinations thereof. In addition, if desired, these compositions may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, stabilizers, or pH buffers.

[0095] Exemplary methods and instructions for administering rhARG1, Co-rhARG1, and Co-rhARG1-PEG (e.g., PEGylated arginine) are provided below. Although the following instructions are specifically directed to PEGylated arginine, these methods and instructions can also be applied to other recombinant arginines 1 and 2.

[0096] [Recommended intravenous dosage regimen:]

[0097] Obtain a baseline plasma arginine concentration before initiating treatment. The recommended initial dose of pegylated arginine for patients with ARG1-D is 0.10 mg / kg, administered as a single intravenous infusion once weekly. If the initial dose of 0.10 mg / kg fails to reduce plasma arginine levels to ≤150 μM / L, the dose can be increased to a maximum of 0.20 mg / kg (once weekly). If plasma arginine levels fall below 50 μM / L during treatment, consider a dose reduction. When administering five or more intravenous doses of pegylated arginine, consider subcutaneous administration of pegylated arginine in patients with ARG1-D and continue to monitor plasma arginine levels periodically.

[0098] [Recommended subcutaneous dosage regimen:]

[0099] When switching from intravenous pegylated arginine therapy to subcutaneous administration, administer the first subcutaneous dose in place of the next scheduled intravenous dose. The initial subcutaneous dose should be the same mg / kg dose as the last intravenous dose administered. The subcutaneous dose may be modified as clinically indicated to ensure plasma arginine levels remain within the range of 50-150 μM / L.

[0100] [Blood arginine monitoring:]

[0101] 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 µmol / L to 150 µmol / L. Thereafter, periodic plasma arginine monitoring is recommended to assess plasma arginine control. Additional plasma arginine monitoring may be necessary when switching to subcutaneous dosing or when dietary changes are made.

[0102] [Preparation and Administration Instructions]

[0103] Pegylated arginase is supplied as a frozen liquid formulation in 10 mL single-use glass vials containing 5 mL of either 1 mg / mL or 5 mg / mL. Each single-use glass vial of pegylated arginase is intended for a single intravenous or subcutaneous injection. Before administration, visually inspect the pegylated arginase for particulate matter and discoloration. Pegylated arginase is a colorless to pale yellow or pale pink solution. Discard if discoloration, turbidity, or particulate matter is present in the vial. Remove the flip-top cap from the vial. Use an alcohol pad to disinfect the rubber stopper of the vial. Use a sterile syringe with an 18G needle to withdraw the appropriate volume of drug from the vial. If more than one vial is required, use a separate needle to withdraw the solution from each vial. Calculate the amount of solution to be withdrawn from the vial for the injection pump. Once the appropriate volume of medication has been drawn into the syringe, immediately withdraw normal saline using a separate needle to achieve a total volume of 40 mL. Calculate the amount of medication to be used as follows: mg / mL Pegylated Arginase Amount = Patient Weight (kg) × Dose Level (mg / kg) 5.0 mg / mL PEGylated arginine enzyme amount = [Patient weight] [(kg) ×] [Dose Level] [(mg / kg)] 5

[0104] Pegylated arginine is administered via intravenous infusion over 30 minutes using an injection pump.

[0105] Table 2: Weight-based dosing for once-weekly 0.1 mg / kg [weight] [(kg)*] [Proposed injection volume] [(mL)] [Vial configuration] [3] [0.30] [1 mg / mL] [4] [0.40] [1 mg / mL] [5] [0.50] [1 mg / mL] [6] [0.60] [1 mg / mL] [7] [0.70] [1 mg / mL] [8] [0.80] [1 mg / mL] [9] [0.90] [1 mg / mL]

[10] [1.0] [1 mg / mL]

[15] [1.5] [1 mg / mL]

[20] [2.0] [1 mg / mL]

[25] [2.5] [1 mg / mL]

[30] [3.0] [1 mg / mL]

[35] [3.5] [1 mg / mL]

[40] [4.0] [1 mg / mL]

[50] [5.0] [1 mg / mL]

[60] [1.20] [5 mg / mL]

[70] [1.40] [5 mg / mL]

[80] [1.60] [5 mg / mL]

[0106] In one or more embodiments, the subcutaneous injection volume has a maximum volume, e.g., a maximum of 2 mL / injection for adult patients and / or a maximum of 1 mL / injection for pediatric patients. If the calculated volume for subcutaneous administration is greater than the maximum volume, a higher vial concentration may be used (e.g., 5 mg / mL instead of 1 mg / mL) and / or the volume may be divided into multiple smaller injections (e.g., a 4 mL injection may be divided into two 2 mL injections).

[0107] [Dosage form and strength]

[0108] Pegylated arginine phosphate injection is a colorless to pale yellow or light pink solution available in 10 mL vials as follows: a. Solution for injection: 5 mL, 1.0 mg / mL b. Solution for injection: 5 mL, 5.0 mg / mL

[0109] [Warnings and Precautions]

[0110] Anaphylactic reactions may occur with the administration of pegylated arginine. Monitor all patients for signs and symptoms of acute allergic reactions (e.g., urticaria, pruritus, erythema, hypotension, tachycardia) during and after pegylated arginine infusion. In the event of a severe anaphylactic reaction, immediately slow or stop pegylated arginine administration and institute appropriate medical care. Consider premedicating patients with a non-sedating antihistamine prior to administration. If corticosteroids are necessary, they should be used with caution due to the potential for hyperammonemia.

[0111] [Pregnancy:Pregnancy Category] [B]

[0112] Reproduction studies have been conducted in mice and rats at doses up to 100 mg / kg. There is no evidence that pegylated arginine harms the fetus. However, there are no adequate and well-controlled studies in pregnant women. Because animal reproduction studies are not always predictive of human responses, pegylated arginine should be used during pregnancy only if clearly needed.

[0113] [Breastfeeding mothers]

[0114] It is not known whether PEGylated arginine is present in human milk. The developmental and health benefits of breastfeeding should be considered along with the mother's clinical need for PEGylated arginine and any potential adverse effects of this drug on the breastfed child.

[0115] [illustrate]

[0116] Pegylated argininase is a cobalt-substituted, recombinant human arginase I covalently coupled to monomethoxypolyethylene glycol (mPEG). It functions by catalyzing the same reaction as argininase 1, converting arginine to ornithine and urea. Human argininase 1 is a binuclear manganese metalloenzyme. To produce pegylated argininase, cobalt is used instead of the manganese cofactor to produce Co-argininase I. Substituting cobalt (Co2+) for native manganese (Mn2+) in the active site of argininase I enhances stability and catalytic activity at physiological pH. Pegylation extends the circulation half-life. The average molecular weight of pegylated argininase is approximately 284 kDa. Pegylated argininase exhibits a specific activity ranging from approximately 320 to 600 units / mg of protein. One unit of activity is defined as the amount of enzyme required to convert 1 μM arginine to ornithine per minute at 37°C.

[0117] Pegylated arginine is intended for intravenous or subcutaneous infusion and is supplied as a sterile, clear, colorless to pale yellow or pale pink solution formulated at concentrations of 1 mg / mL and 5 mg / mL in a buffer containing 50 mM sodium chloride, 5 mM potassium phosphate, and 1.5% w / v glycerol at pH 7.4. It is supplied as a preservative-free, sterile solution in single-use, clear glass vials. Each vial of 1 mg / mL Pegylated arginine drug product contains 5 mL of drug product (5 mg Pegylated arginine / vial). Each vial of 5 mg / mL Pegylated arginine drug product contains 5 mL of drug product (25 mg Pegylated arginine / vial). Vials should be stoppered with a coated rubber stopper and sealed with an aluminum reversible seal. Store frozen at ≤ -60°C and thaw before use.

[0118] [Pharmacodynamics]

[0119] Treatment of adult and pediatric patients with arginase 1 deficiency with pegylated arginase reduces blood arginine concentrations from pre-treatment baseline values to within the normal blood arginine range of 40-115 μM / L. Maximum inhibition of L-arginine levels was observed approximately 8 hours after administration and decreased in a dose-dependent manner, with recovery to pre-dose levels by 168 hours. A strong correlation was observed between pegylated arginase and arginine, with an immediate inhibitory effect on arginine after intravenous administration, and a maximum reduction in arginine concentrations reached within 24 hours of administration.

[0120] [Pharmacokinetics]

[0121] Following intravenous administration in 14 subjects, pharmacokinetic samples were collected throughout the dosing interval from 0 to 168 hours to characterize the relationship between the pharmacokinetics of pegylated arginine and arginine. Pegylated arginine exposure (as measured by Cmax and AUC0-168) increased approximately dose-proportionally across a dose range (0.015 mg / kg to 0.2 mg / kg), with a 13-fold increase in dose resulting in a 14-fold increase in Cmax and AUC0-168. Pegylated arginine accumulation was not observed following the once-weekly intravenous dosing regimen, with a T1 / 2 of approximately 30 hours across this dose range and low to moderate inter-subject variability in exposure parameters (13-46% CV).

[0122] [Animal Toxicology and] [ / ] [or pharmacology]

[0123] The pharmacological effects of pegylated arginase on arginine levels were evaluated in a neonatal transgenic mouse model of arginase I and a tamoxifen-inducible arginase-deficient model in adult mice. These models mimic the human condition in which circulating arginine and arginine metabolites are significantly excessive; however, unlike humans with arginase I deficiency, these animals develop severe and often fatal hyperammonemia. The pharmacological effects were also evaluated in a rat model of arginine-induced hyperargininemia. Pegylated arginase reduced plasma arginine levels in a dose-dependent manner.

[0124] The potential toxicity and TK of PEGylated arginine were evaluated in rats juvenile on postnatal day (PND) 21 (equivalent to 2-year-old humans). Rats were administered weekly intravenous bolus doses of 0.1, 0.3, and 1.0 mg / kg for 6 months, followed by a 6-week recovery period. PEGylated arginine was well tolerated, with no test article-related mortality or significant test article effects observed in the following analyses: food consumption, coagulation, urinalysis, ophthalmoscopy, sexual maturation, growth hormone analysis, bone marrow analysis, Functional Observation Battery (FOB) assessment, and neurobehavioral testing (acoustic startle habituation, locomotor activity, or the Morris water swim maze). No macroscopic findings related to PEGylated arginine were observed at the end of the 6-month period and the 6-week recovery period. Adverse microscopic changes were limited to the testicles and epididymis and were associated with decreased male reproductive organ weights and adverse sperm analysis results at 0.3 mg / kg and 1.0 mg / kg. At 1.0 mg / kg, adverse effects were observed in sperm analysis, including decreased sperm motility, decreased sperm count in the caudal epididymis, decreased sperm concentration, and an increased percentage of abnormal sperm. These observations can be considered directly treatment-related and were associated with microscopic changes of subtle tubular degeneration in the testes at 0.3 mg / kg and 1.0 mg / kg. After a 6-week recovery period, these changes were largely reversible in both the control and 1.0 mg / kg groups, with the exception of an increase in the percentage of abnormal sperm and sperm count. Partial reversibility after 6 weeks is not unexpected, as the normal sperm developmental cycle lasts approximately 9 weeks or longer.

[0125] Importantly, no significant pegylation effects were observed by histopathology. Toxicokinetic data indicate that pegylated arginine exposure was maintained throughout the study. Overall, the NOAEL in females was 1.0 mg / kg. In males, the NOAEL was 0.1 mg / kg based on microscopic changes in the testes at 0.3 mg / kg and 1.0 mg / kg.

[0126] The potential toxicity and TK of pegylated arginase were assessed in cynomolgus monkeys after weekly intravenous bolus administration of 0.1, 0.3, and 1.0 mg / kg for 13 weeks, followed by a 4-week recovery period. Clinical signs observed at 1.0 mg / kg included weight loss, increased incidence of hair loss (whole body), dry / discolored skin (whole body), tremor, loss of appetite, watery stools, decreased activity, ataxia, muscle atrophy, and / or unkempt / hunched appearance. No treatment-related effects were observed in clinical pathology parameters (coagulation, growth hormone, and urinalysis), ECG and ophthalmological examinations, respiratory rate, and blood pressure assessments.

[0127] [supply] [ / ] [Storage and handling methods]

[0128] Pegylated arginase is supplied as a solution for injection.

[0129] Supply PEGylated arginine lysate frozen (≤ -60°C). Diluted PEGylated arginine lysate should be used immediately. If not used immediately, store diluted PEGylated arginine lysate at 2°C to 8°C (36°F to 46°F) for up to 8 hours during administration.

[0130] Examples

[0131] Before describing several exemplary embodiments of the present disclosure, it should be understood that the present disclosure is not limited to the details of construction or process steps set forth in the following description. The present disclosure is capable of other embodiments and of being practiced or carried out in various ways.

[0132] In the following experimental disclosure, the following abbreviations are used: eq (equivalent); M (molar); μM (micromolar); mM (millimolar); N (normality); mol (molecular); mmol (millimolar); 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)

[0133] [Example] [1] [:Cation exchange column chromatography] [(] [Pipeline] [1)]

[0134] In a preferred embodiment, arginase 1 is captured on a cation exchange column (CEX) to reduce product-related and process-related impurities (e.g., host cell proteins (HCPs), DNA, and endotoxins) (see Figure 3 for an overview of the purification process). In a specific embodiment, the first column (column 1) chromatography step in the arginase 1 purification process utilizes SP Sepharose FF resin and an inlet heat exchanger. Column 1 binds arginase 1 using cation exchange chromatography at pH 7.6 in the absence of salt and is eluted using buffers with increasing salt concentrations ( Figure 4(a) ). In one embodiment, the salt is NaCl, and elution from column 1 is performed using 25 mM HEPES, 0.1 M NaCl (pH 7.2-7.6) at room temperature. However, alternative embodiments, such as applying a NaCl gradient to column 1, may be used.

[0135] Figure 4(a) shows a representative purification of arginase 1 on column 1. Approximately three liters of purified E. coli lysate were loaded onto the cation exchange column. As evidenced by the high absorbance at 280 nm, a significant amount of protein was not bound to the column and was detected in the flow-through. The column was then washed with approximately two liters of column wash solution. The arginase 1-enriched fraction (last peak) was then eluted using 0.1 M NaCl, as detected by absorbance at 280 nm.

[0136] [Example] [2] [:Cobalt substitution]

[0137] In a preferred embodiment, the natural manganese coenzyme of arginase 1 is replaced by cobalt. During cobalt replacement (also known as cobalt loading), cobalt ions replace one or both of the two manganese ions normally present in arginase 1. The cobalt replacement step can be performed at a variety of temperatures and cobalt concentrations (see Table 2). Cobalt replacement incubation times can be as short as 10 minutes and can be performed at temperatures above 50°C. In contrast, cobalt loading temperatures can be as low as 1°C or 5°C and performed for over 8 hours. Furthermore, higher proportions of cobalt loaded into arginase 1 can result in higher specific activity.

[0138] Arginase 1 eluted from column 1 (also referred to as column 1 pool) can be kept at room temperature for the cobalt replacement step. In one embodiment, the Column 1 pool is diluted 50-fold by adding a cobalt chloride stock solution (0.5 M CoCl₂) to the Column 1 pool at a defined rate, resulting in a final cobalt chloride concentration of 10 mM. The cobalt replacement solution is then mixed at 20°C for two hours. In another embodiment, cobalt loading of arginase 1 is performed in a 10 mM CoCl₂ solution at room temperature for approximately 2 to 8 hours.

[0139] An overview of the cobalt loading steps can be found in Table 3.

[0140] Table 3

[0141] [Example] [3] Ultrafiltration [ / ] [percolation] [1 (UF / DF 1)]

[0142] UF / DF 1 removes free cobalt ions and exchanges the Co-arginase 1 into a solution prepared for anion exchange chromatography. The UF / DF 1 step utilizes a membrane with a molecular weight cutoff of 30 kDa. A key function of this step is to reduce free cobalt levels and perform buffer exchange on the Co-arginase 1 pool prior to anion exchange chromatography. The membrane is sanitized with a cleaning solution (0.5 N NaOH) and rinsed with water. A normalized water permeability test (NWP) is performed, followed by equilibration and subsequent use in production. Once the UF / DF system is equilibrated, the Co-arginase 1 pool is immediately diafiltered using three diafiltration volumes of 25 mM HEPES, 0.1 M NaCl (pH 7.6), followed by four diafiltration volumes of 50 mM Tris (pH 8.4). Following diafiltration, the pool is recirculated and recovered from the system using twice the system volume with 50 mM Tris (pH 8.4).

[0143] UF / DF1 membranes are cleaned by performing a 2 M NaCl rinse, followed by a denaturation wash step using 0.5 N NaOH and recirculating for 30 minutes. The system is rinsed with purified water and a NWP test is performed to evaluate the effectiveness of the cleaning procedure. The membranes can be stored in 0.1 N NaOH.

[0144] In an alternative embodiment, the buffer is first exchanged to 25 mM HEPES, 0.1 M NaCl (pH 7.2-7.6) and then to 50 mM Tris (pH 8.1-8.5).

[0145] [Example] [4] [:Anion column chromatography] [(] [Pipeline] [2)]

[0146] A preferred embodiment of arginase 1 purification utilizes another column, an anion exchange column chromatography ("Column 2"). One embodiment of Column 2 is Q Sepharose FF resin. One function of this Column 2 step is to reduce process-related impurities (e.g., host cell DNA and endotoxins from the UF / DF1 pool). Column 2 binds these impurities, while Co-arginase 1 flows through it and is collected in the column flow-through during the loading and wash steps. In one embodiment, the anion exchange flow-through chromatography of Column 2 is performed using Q Sepharose FF, and up to 40 g of protein / L of resin is loaded onto the column using 50 mM Tris (pH 8.1-8.5) buffer.

[0147] In another embodiment of these methods, the first protein product is loaded onto an anion exchange column to capture impurities, while arginase 1 is recovered in the flowthrough. Figure 4(b) shows a representative chromatogram of the purification of arginase 1 on an anion exchange column (column 2). As can be seen from the absorbance at 280 nm, a significant amount of protein is detected in the flowthrough. Impurities are captured on column 2 and do not elute into the column 2 pool (also referred to as the second protein product), which is further enriched for arginase 1.

[0148] [Example] [5] [:] [Capto] [Multicomponent column chromatography] [(] [Pipeline] [3)]

[0149] In a preferred embodiment, the arginase purification process utilizes a third chromatography column (column 3). In one embodiment, column 3 is a Capto multicomponent chromatography (MMC) column or, alternatively, a size-exclusion column. In embodiments utilizing MMC, arginase 1 is captured on the column, while process-related impurities (e.g., host cell proteins (HCPs), DNA, and endotoxins) are washed out in the flow-through. In this embodiment, Co-arginase 1 can be captured by the column at pH 8.4 in the absence of salt and then eluted using a buffer with increasing salt concentration. A representative example of a Capto multicomponent cation exchange chromatography column is shown in Figure 4(c).

[0150] In one embodiment, MMC chromatography (column 3) uses approximately 15 column volumes to load up to 30 g of protein / L of resin, and elution is performed using a high salt step with 50 mM Tris, 250 mM NaCl (pH 8.1-8.5). In several embodiments, the flow-through from the anion exchange column (column 2) is loaded onto a Capto MMC column at pH 8.4, washed, and then bound Co-arginase 1 is eluted using 50 mM tromethamine, 250 mM NaCl.

[0151] [Example] [6] Ultrafiltration [ / ] [percolation] [2 (UF / DF 2)]

[0152] UF / DF 2 concentrates arginase 1 and exchanges the protein for a pre-PEGylated intermediate. The UF / DF 2 step utilizes a membrane with a molecular weight cutoff of 30 kDa. A key function of this step is to buffer-exchange the column 3 pool containing the unPEGylated Co-arginase 1 intermediate prior to PEGylation (or prior to additional filtration and storage). The membrane is sanitized with a cleaning solution (0.5 N NaOH) and rinsed with water. Once the UF / DF system is equilibrated, the column 3 pool (also referred to as the third protein product) is immediately diafiltered using five diafiltration volumes of 20 mM sodium phosphate, 50 mM sodium chloride, 1.5% (w / v) glycerol, pH 7.4. If the column 3 pool concentration is < 8 g / L, the pool is further concentrated to 8 g / L. After diafiltration (and, if necessary, concentration), the pool is recirculated and recovered from the system using twice the system volume with 20 mM sodium phosphate, 50 mM sodium chloride, 1.5% (w / v) glycerol (pH 7.4). Following recovery, a two-step dilution can be performed using the diafiltration solution. The first dilution has a target concentration of 6 g / L, and the second step has a target concentration of 5 g / L. This target can be achieved using both steps. If the concentration after the first dilution is within the target range, the second step may not be necessary.

[0153] [Example] [7] [:Intermediate Filtration and] [UF / DF 3]

[0154] Prior to the PEGylation reaction, the cobalt-containing arginase 1 can be stored for an extended period (e.g., frozen for an extended period). The intermediate Co-argininase can be filtered through a 0.2 μm filter and frozen for long-term storage.

[0155] The UF / DF3 step uses a membrane with a 30 kDa molecular weight cutoff. One function of this step is to buffer exchange and concentrate the filtered UF / DF2 pool (fresh or thawed) to provide optimal conditions for PEGylation. If using frozen Co-argininase 1 intermediate as starting material, it should be thawed at room temperature for up to 36 hours. The membrane is sanitized with a cleaning solution (0.5 N NaOH) and rinsed with water. A normalized water permeability test (NWP) is performed, followed by equilibration and subsequent use in production. Once the UF / DF system is equilibrated, the Co-argininase 1 intermediate is immediately diafiltered using five diafiltration volumes of 0.1 M sodium phosphate (pH 8.4). Following diafiltration, the pool is concentrated, recirculated, and recovered from the system using twice the system volume with 0.1 M sodium phosphate (pH 8.4). Following recovery, a two-step dilution can be performed using the diafiltration solution. The first dilution has a target concentration of 11 g / L, and the second step has a target concentration of 10 g / L. Using two steps facilitates achieving the target level. If the concentration after the first dilution is within the target range, the second step may not be necessary.

[0156] For UF / DF 2 and UF / DF 3 steps, the first buffer exchange can be to 20 mM sodium phosphate, 50 mM NaCl, 1.5% glycerol (pH 7.4, ≥5 DV), and the protein can be concentrated to approximately 5.0 mg / mL. The second buffer exchange can be to 0.1 M sodium phosphate (pH 8.1-8.5), and the protein can be concentrated to approximately 10.0 mg / mL (in preparation for PEGylation of the drug substance).

[0157] [Example] [8] [:Arginase] [1] [PEGylation]

[0158] PEGylation covalently attaches PEG (polyethylene glycol) to the Co-argininase 1 (drug substance) molecule (see Table 4 for representative examples of the PEGylation step). In one example, the PEGylation reaction covalently binds a 5000 Da PEG molecule to Co-argininase 1. In alternative embodiments, PEGylation can be performed prior to cobalt substitution of argininase 1 or at other points during the production process. In one example, the PEG conjugation reaction can be performed using solid or liquid methoxyPEG succinimidyl carboxymethyl ester to react with sterically available lysine on Co-argininase 1. The resulting PEGylated protein (Co-rhARG1-PEG) has a molecular weight of approximately 280 kDa. The PEGylated pool can be filtered and stored at 2-8°C until UF / DF4 operation.

[0159] Table 4: PEGylation Process for Co-rhARG1 Drug Substance [step] [Process Operation] 1 Thaw intermediates (if applicable) ↓ 2 Ultrafiltration / diafiltration ↓ 3 Add solid methoxy PEG succinimidyl carboxymethyl ester (MW 5000) and incubate for >15 min pH 8.4 ↓ 4 Ultrafiltration / diafiltration ↓ 5 Filter the (Co-rhARG1-PEG) drug substance, fill into polycarbonate bottles, and freeze at ≤ -60°C

[0160] In one embodiment, solid methoxy PEG succinimidyl carboxymethyl ester (MW 5000) can be added to a solution containing arginase 1 at a 19.3x molar excess and incubated at pH 8.4 for 0.5-4.0 hours.

[0161] After PEGylation, ultrafiltration / diafiltration removes unbound PEG, exchanges arginase 1 into formulation buffer, and concentrates arginase 1 for the formulation step. This UF / DF4 step utilizes a membrane with a 100 kDa molecular weight cutoff. One function of this step is to buffer-exchange the PEG pool into the final formulation while simultaneously removing free PEG. The membrane used for this purpose is sanitized with a cleaning solution (0.5 N NaOH) and rinsed with water. Once the UF / DF system is equilibrated, the PEG pool is immediately diafiltered using ten diafiltration volumes of 5 mM potassium phosphate, 50 mM sodium chloride, 1.5% (w / v) glycerol, pH 7.4. Following diafiltration, the pool is withdrawn from the system under pressure. The recovered UF / DF4 pool was diluted to 5 g / L using 5 mM potassium phosphate, 50 mM sodium chloride, 1.5% (w / v) glycerol (pH 7.4) before final filtration and filling. In an alternative embodiment, arginase 1 was exchanged into 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.

[0162] In certain embodiments, a formulation buffer (pH 7.4) containing 5 mM potassium phosphate, 50 mM sodium chloride, and 1.5% glycerol was found to enhance 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 K 2 HPO 4 and 4 mM KH 2 PO 4.

[0163] The drug substance (Co-rhARG1-PEG) is a PEGylated, cobalt-substituted human arginase 1 prepared by conjugating an activated PEG molecule to the ε-amino group of lysine and the amino group of the N-terminal amino acid. The molar ratio of PEG molecules / protein is determined using a dye-based fluorescence assay using o-phthalaldehyde. O-phthalaldehyde reacts specifically with primary amines in the presence of thiols to form fluorescent derivatives. Measurement of the fluorescent signal allows quantification of the reactive free amines present in the protein molecule. Quantification is based on a standard curve using N-acetyl lysine. The number of PEGylated amines per protein can be determined by subtracting the number of free amines measured by fluorescence analysis of the PEGylated drug substance from the theoretical number of free amines present in unconjugated Co-arginase 1. The theoretical number of free amines from lysine residues and the N-terminal amino acid is 25. Free unconjugated PEG in the drug substance was measured by SEC-HPLC, with detection based on the refractive index. Results were expressed as µg / mL free PEG (see Table 5).

[0164] Table 5: SEC-HPLC method parameters / free PEG / Co-rhARG1-PEG drug substance [illustrate] [method parameters] Pipeline TOSOH G3000PWxl, 7.8 × 300 mm, 7µm column detector Refractive index detector Injection volume 10 µL flow rate 0.8 mL / min Automatic sampler temperature 5℃ Column temperature 20℃ Mobile phase A 1X PBS Stop Time 20 minutes

[0165] [Example] [9] [:Drug intermediates] [CIEX-HPLC] [Representation]

[0166] During E. coli fermentation, various charge variants of arginase 1 are produced. These charge variants can be analyzed by cation exchange HPLC (CIEX-HPLC) using a TSK gel cation exchange column. The analysis conditions used were: 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 minutes; column temperature: 22°C; and the mobile phase gradient shown in Table 6.

[0167] Table 6: CIEX-HPLC charge variant Co-arginase 1 intermediate gradient program [minute] [Mobile phase] [(B) %] 0.00 0 1.95 0 17.00 30 23.00 60 25.00 100 30.00 100 30.10 0 40.00 0

[0168] Samples were diluted with preparation buffer prior to analysis. Results are presented as percentage charge variant distribution. A representative chromatogram is shown in Figure 5(a), where six major peaks of the Co-argininase 1 intermediate were typically observed.

[0169] [Example]

[10] [:drug substance] [iCIEF] [Representation]

[0170] A pharmaceutical intermediate was PEGylated to form a drug substance. Compared to other examples developed as part of this invention, PEGylation of the drug intermediate made the CIEX-HPLC method less suitable for the drug intermediate. Anion IEX-HPLC was evaluated but was not adequately resolved. Alternatively, an imaging capillary isoelectric focusing (iCIEF) method was developed to analyze charge variants of the drug substance.

[0171] In imaged capillary isoelectric focusing (iCIEF), analytes migrate through the capillary via the counter-migration of hydronium ions (anolyte) and hydroxyl ions (catholyte) under an applied electric field. The sample is diluted in a matrix containing carrier ampholytes and pI markers. Proteins are separated in two focusing steps. An initial prefocusing step establishes the pH gradient. During the second, higher-voltage focusing step, charge variants are more sharply focused and separated. UV absorbance images of the entire capillary are digitally captured every 30 seconds and after the focusing step is complete.

[0172] Results are expressed as percentage charge variant distribution. A representative electropherogram is shown in Figure 5(b), where nine major peaks of the drug substance are observed. Peaks 3 and 4 are clustered together, as the resolution between these peaks appears variable. The relative areas of these peaks are provided in Table 7.

[0173] Table 7: iCIEF characterization of charge variants of Co-rhARG1-PEG [name] [pI] [area] [area] [%] 1 markers 4.220 2 Peak 1 6.111 130195 12 3 Peak 2 6.268 245568 twenty two 6 Peak 3+4 6.360 255343 twenty three 7 Peak 5 6.501 226011 20 8 Peak 6 6.604 156066 14 9 Peak 7 6.708 71805 6 10 Peak 8 6.794 27780 2 11 Peak 9 6.866 8847 1 12 markers 7.650

[0174] [Example]

[11] [:] [Co-] [Arginase] [1] [Enzyme activity of intermediates and drug substances]

[0175] An enzymatic assay used to measure the activity and characterize the Co-argininase 1 intermediate and the Co-rhARG1-PEG drug substance monitors the conversion of arginine to ornithine. One enzyme concentration in the reaction mixture was tested at seven different arginine substrate concentrations ranging from 0 to 2 mM. Reactions were performed at 37°C for a fixed time. The reaction time was established to ensure less than 10% substrate consumption at any given substrate concentration. The reaction was terminated, and the ornithine product was derivatized and quantified by reverse-phase UPLC.

[0176] Examples of plots of reaction rate versus substrate concentration, along with representative Kcat, Km, and Kcat / Km values, are shown in FIG8(a) (Co-argininase 1 intermediate) and FIG8(b) (Co-rhARG1-PEG drug substance).

[0177] [Example]

[12] [:Analysis of Cobalt and Manganese]

[0178] Cobalt, residual manganese, and free cobalt were measured using inductively coupled plasma mass spectrometry (ICP-MS). Samples were digested by microwave heating with 1% nitric acid and 6% hydrogen peroxide to release all metals from the matrix. The resulting digests were analyzed by ICP-MS. Cobalt and residual manganese samples were digested without any sample treatment. Free cobalt was measured in permeate samples that had been ultrafiltered to separate the enzyme from the permeate to measure cobalt not associated with the enzyme. Table 8 summarizes certain properties of Co-arginase 1 intermediates.

[0179] Table 8: Typical Co-arginine enzyme 1 intermediate characteristics test Purpose Analysis Principle Typical values CIEX HPLC Charge HPLC Peak 1: 0-4% Peak 2: 2-8% Peak 3: 10-20% Peak 4: 16-30% Peak 5: 40-52% Peak 6: 4-15% SDS-PAGE (reducing) purity Charge mobility ≥95% K m k cat active HPLC 0.15-0.24 mM 200-380 / sec Specific activity active HPLC 320-600 U / mg cobalt active ICP-MS 7-13 µg / mL Free cobalt stability ICP-MS ≤0.05 µg / mL Residual manganese Purity, activity ICP-MS ≤1 µg / mL pH acidity pH 7.0-7.8

[0180] Table 9: Typical Co-rhARG1-PEG Drug Substance Properties test Purpose Analysis Principle Typical values Charge heterogeneity quality Imaging capillary isoelectric focusing (iCIEF) Peak 1: <20% Peak 2: <30% Peaks 3 and 4: 10-30% Peak 5: 15-30% Peak 6: 10-25% Peak 7: <25% Peak 8: <15% Peak 9: <8% K m k cat active HPLC 0.15-0.24 mM 200-380 / sec Specific activity active HPLC 320-600 U / mg Total cobalt active ICP-MS 7-13 µg / mL Residual manganese active ICP-MS <1 µg / mL Free cobalt stability ICP-MS ≤0.05 µg / mL PEG:protein molar ratio PEGylation HPLC 8-16 mol / mol Free PEG impurities HPLC <500 µg / mL pH* acidity pH 7.0-7.8

[0181] [Example]

[13] [:] [Co-] [Arginase] [1] [Post-translational modification of intermediates]

[0182] Various techniques (eg, peptide mapping, LC-MS intact mass spectrometry) and reversed-phase LC / MS were used to detect post-translational modifications of Co-argininase 1 intermediates. A summary of all identified modifications is listed in Table 10.

[0183] Table 10: Identified Co-arginase 1 intermediate modifications [modification] [Detection method] N-terminal glucuronidation Peptide mapping (Glu-C digestion) RP-HPLC-MS / MS and LC-MS RP-UPLC / MS Phosphogluconylation RP-UPLC / MS Di-gluconic acidification RP-UPLC / MS N-terminal methionine truncation Peptide mapping (Glu-C digestion) RP-HPLC-MS / MS and LC-MS Acetylation Peptide mapping (Glu-C digestion) RP-HPLC-MS / MS and LC-MS N-deamination / succinimide formation Peptide mapping (Glu-C digestion) RP-HPLC-MS / MS and LC-MS

[0184] Characterization revealed that, when modifications of the Co-arginase 1 intermediate were present, the predominant modification was N-terminal glucuronidation (confirmed by peptide mapping). Additional characterization of the modified Co-arginase 1 species was performed by testing samples obtained at three time points: fermentation, post-column 1, and for the drug intermediate from column 3. The analytical method typically requires dissociation of the arginase and analysis as a monomer. Argininase 1 N-terminal glucuronidation (analyzed as a monomer) typically ranged from 10.8% to 13.9%. Other modifications observed in samples from the three time points were N-terminal phosphogluconylation monomers (4.3% to 6.5%) and di-glucuronidation monomers (0.7% to 1.2%). In samples used for normalization reference production experiments, levels of unmodified Co-arginase 1 (monomer) and the Co-arginase 1 intermediate were comparable, at 80.6% and 83.6%, respectively. Standard conditions used for the purification process (ie, no salt gradient applied to column 1) modestly altered the relative levels of unmodified monomer and the Co-arginase 1 intermediate (81.1% and 83.6%).

[0185] Table 11: LC / MS results for characterization of Co-argininase 1 [Sample Number] [Arginase] [1] [monomer] [(] [Unmodified] [) (%)] [Glucolylarginase] [1] [monomer] [(%)] [Argininase phosphogluconylase] [1] [monomer] [(%)] [two] [-] [Glucosylarginase] [1] [monomer] [(%)] Standardized reference 80.6 13.9 4.3 1.1 Fermentation 81.1 12.4 6.5 1.2 After the string 81.4 12.2 5.4 0.9 Drug substances 83.6 10.8 4.9 0.7

[0186] [Example]

[14] [:pipeline] [1] [Changes in conditions]

[0187] In an alternative embodiment, a NaCl gradient can be applied to column 1. Using a NaCl gradient on column 1 enables separation of different arginase 1 variants to select preferred embodiments. Figure 7 shows a 0.0-0.2 M NaCl gradient applied to column 1. Individual fractions collected from column 1 were analyzed by SE-HPLC, CEX-HPLC, and RP-HPLC.

[0188] An analytical CEX-HPLC method was used, which assigned peak numbers 1 to 6 to the charge variants of arginase 1 (see Figure 5(c)). The peak numbers correspond to the various gluconylation states, as well as to ungluconylated arginase 1. This analysis revealed six peaks in arginase 1 eluted with a NaCl gradient. Arginase 1 variants eluting early in the column 1 elution peak were assigned peak numbers 1, 2, and 3. Peak 4 eluted in the highest concentration of eluted arginase 1, while peaks 5 (unmodified arginase 1) and 6 eluted later in the elution peak. Therefore, 0.0-0.2 M NaCl successfully separated the different charge variants of arginase 1.

[0189] Alternative NaCl gradients can be used for column 1 elution, such as 0-0.5 M NaCl. It was found that the use of a NaCl gradient can reproducibly separate arginase 1 into six distinct peaks, thereby enabling the selection of specific arginase 1 variants for further processing to manufacture drug substances or drug products.

[0190] The first protein product (and the arginase 1 variant) was further analyzed by LC / MS (see Figure 6). LC / MS analysis identified the specific glucuronidation species produced by arginase 1 production in E. coli. LC / MS analysis identified unmodified arginase 1, glucuronidated arginine 1, phosphogluconated arginine 1, and 2X glucuronidated arginine 1.

[0191] Table 12 shows that applying a 0-0.2 M NaCl gradient (and corresponding fractionated CEX peaks 1-6) produces fractions with varying degrees of glucuronidation. Each of peaks 1-6 was analyzed by LC / MS. The data show that the major peak (peak 5) has a high percentage of unglucuronidated arginase 1 and a high specific activity. Depending on the desired properties, different fractions (corresponding to peaks 1-6) can be collected for further processing.

[0192] Table 12: LC / MS analysis of drug intermediate peaks 1-6. fractions Concentration (mg / mL) Mass % of unglucosaminidase 1 monomer determined by MS Specific activity (U / mg) Peak 1 0.2 28.4 118 Peak 2 0.9 37.8 286 Peak 3 3.4 56.2 298 Peak 4 6.5 68.2 349 Peak 5 12.0 96.6 329 Peak 6 2.2 97.1 198

[0193] In addition to varying NaCl concentrations on column 1, varying amounts of protein can also be loaded onto column 1 to enhance the purification of ungluconylated arginase 1 material.

[0194] Changing the loading factor of column 1 and using a NaCl gradient on column 1 compensated for unexpected perturbations that occurred during the fermentation of E. coli producing the glucuronidase 1 species.

[0195] [Example]

[15] [:Changes in fermentation conditions]

[0196] Experiments were conducted to determine the stability of fermentation conditions used to produce arginase 1. Table 13 shows that fermentation of E. coli at a suboptimal pH of 7.6 resulted in a greater degree of gluconylation compared to the optimal pH of 7.2. Vessels B1, B8, and B12 used the following optimal fermentation conditions: pH 7.2, 30% dissolved oxygen, and a 0.06 mL / min medium feed rate. Vessel B3 was used to ferment E. coli expressing arginase 1 at pH 7.6 (above the optimal pH). Increasing the pH increased the proportion of phospho-gluconylation adducts (23% vs. 10-12% in the control).

[0197] Table 13: Glucolylated Argininase 1 Observed in Fermentation Vessels container Natural Arg 1 Gluconylation Phospho-glucuronidation 2X Gluconolactone Gluconylation and phospho-gluconylation B1 88.5 6.2 5.3 - -- B8 89.2 6.3 4.6 -- -- B12 89.0 5.9 5.1 -- -- average value 88.9 6.1 5.0 -- -- B3 76.7 15.0 5.8 1.8 0.6

[0198] [Example]

[16] [:pipeline] [1] [Changes in the load factor above]

[0199] Different amounts of E. coli cell lysate were applied to column 1 to determine the effect on the purification of arginase 1 charge variants, as well as the yield and purity. Loading factors ranging from 15 to 60 g protein / L resin were used under various conditions to demonstrate the variation in CIEX charge species characteristics (Table 14). Higher loading factors resulted in better separation of gluconylated variants (although this may compromise yield depending on the fractions collected). For example, using a loading factor of 20 mg protein / mL resin, Peak 5 was 45.8%, while this value increased to 50.0% at a loading factor of 40 mg / mL.

[0200] Table 14: Effect of column 1 loading factor on protein product 1

[0201] [Example]

[17] [:] [1 / 2] [Clinical study]

[0202] The drug product produced by the methods of the present invention was used in a Phase 1 / 2, open-label study to evaluate the administration of Co-rhARG1-PEG in individuals with arginase 1 deficiency and hyperargininemia. The primary objective of this study was to evaluate the safety and tolerability of intravenous (IV) administration of Co-rhARG1-PEG in individuals with hyperargininemia / arginase 1 deficiency. Secondary objectives were to determine the effect of IV-administered study drug on plasma arginine concentrations; to determine the effect of IV-administered study drug on plasma guanidinium compounds (GC); and to characterize the pharmacokinetic (PK) profile of IV-administered study drug. Additional objectives included evaluation of clinical outcome measures used to capture clinical benefit, such as the 6-Minute Walk Test (6MWT), Gross Motor Function Measure (GMFM) parts D and E, and the Adaptive Behavior Assessment System (ABAS).

[0203] Phase 1 / 2 data demonstrated that Co-rhARG1-PEG effectively and sustainably lowers plasma arginine. Furthermore, controlling plasma arginine levels in patients was associated with clinically beneficial responses in motor and adaptive behavior. Treatment was generally well tolerated. Allergic reactions were rare and managed with standard measures.

[0204] The Co-rhARG1-PEG drug product used in this study was supplied as a liquid formulation in 10 mL single-use glass vials containing 5 mL of formulated drug product at a concentration of 1 mg / mL. The drug was formulated in 50 mM NaCl, 1 mM K2HPO4, 4 mM KH2PO4, and 1.5% w / v glycerol.

[0205] The Phase 1 / 2 study was conducted in two parts: Part 1 (single ascending dose escalation) and Part 2 (repeat dosing). The study design for the open-label extension of Phase 1 / 2 trials 101A and 102A is shown in the figure below:

[0206] Part 1 introduced patients to the drug and focused on safety. Part 2 was designed to maintain patients on a consistent dose and to observe markers of clinical effectiveness. Baseline arginine levels were assessed before each part. All patients who participated in Part 1 continued arginase 1 in Part 2, provided they were eligible for continued treatment.

[0207] In the study, each patient received an escalating starting dose in Part 1, with a 2-week washout / observation period between each successive dose level. Potential doses for each patient in Part 1 were 0.015, 0.03, 0.06, 0.10, 0.15, 0.20, and 0.30 mg / kg, with 2-week intervals as needed to optimize plasma arginine. Any given dose could be repeated, or the dose could be increased / decreased between designated dose levels if emerging data from previous dose levels met certain criteria. For example, dose escalation could be discontinued if one or more of the following stopping criteria were met: patient plasma arginine levels < 40 μM in all samples collected for at least 40 consecutive hours (± 2) after dosing; or mean patient plasma arginine levels < 115 μM in all samples collected for at least 112 consecutive hours (± 2) after dosing.

[0208] If these events do not occur, the patient can be escalated to the next higher dose level of arginase 1 every 2 weeks until any dose escalation criterion is met or the patient has received the highest dose on this 0.30 mg / kg regimen. Ultimately, the dose can be increased to above 0.30 mg / kg for therapeutic purposes.

[0209] Part 2 is a repeat dosing phase for patients who have completed Part 1. Part 2 aims to identify a dose and regimen for each patient that safely optimizes plasma arginine within the range of approximately 40 μM to approximately 115 μM during repeated dosing, with an emphasis on maintaining pre-dose levels below 150-200 μM. If data indicate that a dose-response effect can be optimally investigated during repeated dosing, several dose levels may be used in Part 2. Arginine levels during treatment will also be compared to pre-treatment arginine levels.

[0210] Patients who completed Part 2 of 101A were eligible to participate in the long-term open-label extension (OLE) trial (NCT03378531). Treatment was initiated with 24 weekly intravenous doses, with an optional switch to subcutaneous administration for the remainder of the 3-year OLE period.

[0211] result

[0212] Across all patients, mean Cmax and mean AUC0-168 increased dose-proportionally. For Co-rhARG1-PEG dose levels of 0.015, 0.03, 0.06, 0.1, and 0.2 mg / kg, mean (± SD) Cmax were 0.428 ± 0.0915, 0.723 ± 0.247, 1.73 ± 0.538, 2.27 ± 0.238, and 6.13 (N=1) µg / mL, respectively. Anti-drug antibodies (ADAs) had a minimal effect on mean Cmax in both ADA-positive and ADA-negative patients (Figure 9).

[0213] AUC (AUC 0-168, AUC 0-∞) showed dose-proportional changes across the dose range studied, with the notable absence of significant changes between 0.06 mg / kg and 0.1 mg / kg (using available data). The estimated mean clearance (CL) ranged from 0.789 mL / hr / kg to 1.57 mL / hr / kg for all patients and from 0.776 mL / hr / kg to 1.33 mL / hr / kg for ADA-negative patients. The estimated mean volume of distribution (Vss) ranged from 35.3 mL / kg to 52.1 mL / kg for all patients and from 32.8 mL / kg to 52.1 mL / kg for ADA-negative patients.

[0214] Part 1 of the study facilitated the selection of the optimal (individual) starting dose for each patient in Part 2 using the observed PD (arginine) response. During Week 1 of Part 2, mean circulating drug concentrations in all patients trended upward with increasing doses of Co-rhARG1-PEG across the evaluated dose range. Following the first dose of Co-rhARG1-PEG in Part 2, mean Cmax increased in all patients in a dose-proportional manner. For Co-rhARG1-PEG dose levels of 0.015, 0.03, 0.04, 0.06, 0.09, 0.1, and 0.12 mg / kg, the mean (± SD) Cmax were 0.292 (N=1), 0.395 (N=1), 1.01 ± 0.221, 1.75 ± 0.391, 1.99 (N=1), 2.34 (N=1), and 2.87 ± 0.626 µg / mL, respectively.

[0215] In Part 2, mean circulating drug concentrations generally increased with increasing doses of Co-rhARG1-PEG at Week 8 in all patients. At Week 8, there was no significant ADA effect at the PK-useful concentrations. Based on these data, it is assumed that steady-state was achieved in the majority (13 of 14) of patients at this time. Following the 8th quarterly dose of Co-rhARG1-PEG, mean Cmax and AUC0-168 were dose-proportional in all patients.

[0216] In addition to pharmacokinetic data, pharmacodynamic (arginine) data were also collected (Figure 10). In Part 2, patients with arginase 1 deficiency were administered intravenous doses of Co-rhARG1-PEG every week (qw), with the starting dose selected based on the observed PD (arginine) response in Part 1. Following the first Qw intravenous dose of Co-rhARG1-PEG, circulating arginine levels did not decrease significantly, particularly for doses of 0.04 mg / kg or higher. In some cases, individual arginine concentrations fell below 40 µM. Furthermore, at doses ≥ 0.04 mg / kg and immediately prior to administration of the second Qw (qw) dose of Co-rhARG1-PEG, the majority of patients did not fully recover to their starting arginine levels.

[0217] In summary, Co-rhARG1-PEG exposure generally increased with increasing doses, and arginine inhibition also increased. Individualized dose optimization was performed in Part 1, resulting in varying numbers of patients at each dose level across a range of doses during Weeks 1 and 8 of Part 2.

[0218] [Example]

[18] [:Subcutaneous administration]

[0219] After completion of Part 2 of the Phase 1 / 2 study in Example 17, some patients were switched from intravenous to subcutaneous administration of Co-rhARG1-PEG. Surprisingly, subcutaneous administration of Co-rhARG1-PEG resulted in a pharmacodynamic profile superior to that of intravenous administration. Also surprising was that the same formulation used for intravenous administration could be successfully used for subcutaneous administration of Co-rhARG1-PEG.

[0220] Compared to intravenous administration, subcutaneous administration of Co-rhARG1-PEG maintained patient arginine levels within the optimal (healthy) target range for plasma arginine concentrations for a longer period of time (Figure 11). Optimal optimized plasma arginine concentrations in patients range from approximately 40 μM to approximately 115 μM (during repeated dosing), with an emphasis on maintaining pre-dose levels below 150-200 μM. As can be seen in Figure 11, subcutaneous administration of Co-rhARG1-PEG resulted in arginine concentrations above the lower limit of 40 μM and below the upper limit of 115 μM. Surprisingly, subcutaneous administration resulted in arginine concentrations well within the optimal range. This suggests that patients will remain within the appropriate plasma arginine concentration range until receiving another weekly dose of Co-rhARG1-PEG.

[0221] [Example]

[19] [:] [1 / 2] [Pharmacodynamics and clinical responses in phase 1 clinical studies and open-label extension]

[0222] Sixteen patients (11 pediatric and 5 adult) were enrolled in Part 1 of 101A, and 15 entered Part 2 of 101A. Two patients withdrew from the trial for personal reasons (one patient after Dose 3 in Part 1 and one patient after Dose 3 in Part 2). All 14 patients who completed Part 2 of 101A entered the OLE trial.

[0223] The baseline characteristics of the patients are shown in Table 15.

[0224] Table 15: Baseline characteristics Age, years, median (range) 15 (5-31) Female, n (%) 11 (69) Arginine, µM, median (range) 389 (238-566) Ammonia, µM, median (range) 38 (9-77) ALT (U / L), median (range) 34 (15-171) ARG1 mutation, n (%) homozygous Compound hybrid 11 (68.8) 5 (31.2)

[0225] Analysis of plasma arginine and guanidino compounds levels revealed a significant and sustained decrease in plasma arginine levels (Figure 12(a) shows a median decrease of 274 µM from baseline after 20 doses of PEG-arginine). The decrease in plasma arginine from baseline to doses 1, 8, and OLE was statistically significant (p<0.001). The decrease in plasma arginine was accompanied by a decrease in plasma guanidino compounds (GC). Figure 12(b) shows the decrease in plasma levels of guanidinoacetic acid (GAA), N-α-acetyl-L-arginine (NAA), α-keto-δ-guanidinovaleric acid (GVA), and arginine (ARGA) at baseline and during the OLE.

[0226] Fifteen of 16 patients completed all motor assessments at baseline (patient 13 was confined to a wheelchair) (Figure 13(a)). Deficits were defined as follows: 6MWT: less than the lower 5%; GMFM Part D: <35 / 39; GMFM Part E: <68 / 72; ABAS-3: ≤85. 88% (14 / 16) of patients had at least one motor deficit at baseline; 88%, 50%, and 56% of the 16 patients were classified as having baseline deficits on the 6MWT, GMFM Part D, and GMFM Part E, respectively. The ABAS-3 assessment of adaptive behavior was available for 10 patients at baseline. Six patients could not be tested for technical reasons, including language limitations, age, and cognitive impairment. Eight of 10 patients (80%) had baseline deficits in one or more domains assessed by the ABAS-3.

[0227] Overall clinical responses demonstrated that 11 / 14 patients (79%) were classified as responders at Dose 20 based on an improvement of ≥1 MCID in at least one of the 6MWT, GMFM-D, or GMFM-E assessments (Figure 13(b)). The Dose 20 data demonstrated that the 6MWT, GMFM-D, and GMFM-E were sufficiently sensitive to capture changes in clinical benefit in patients with ARG1-D. The percentage of overall responders increased significantly from Dose 8 to Dose 20. All five patients (100%) who reached Dose 44 maintained their Dose 20 overall clinical response status as responders.

[0228] All responders to individual components achieved an improvement of ≥1 MCID (Figure 13(b) and Figure 14). For the 6MWT, 7 / 13 (54%) patients were responders to this individual component. The mean 6MWT change was 32 meters for all patients and 66 meters for the 7 responders. For GMFM-D, 5 / 8 (63%) patients with baseline deficits were responders to this individual component (mean MCID: 1.84, range: 1.21 to 3.33). For GMFM-E, 5 / 8 (63%) patients with baseline deficits were responders to this individual component (mean MCID: 4.79, range: 1.67 to 8.33). The percentage of responders for individual motor components was significantly greater at dose 20 than at dose 8.

[0229] Data from all patients after 20 doses of pegarginase demonstrated significant and sustained reductions in plasma arginine, improvements in key disease manifestations, and a clinical response rate of 79%. The Phase 1 / 2 and OLE trials confirmed the value of using the 6MWT, GMFM-D, or GMFM-E as tools to capture the clinical benefit of pegarginase. Pegarginase was well tolerated, and the rate of treatment-related adverse events decreased over time. The improved arginine control and evidence of clinical benefit following pegarginase treatment further validate the key endpoints and design elements of the pivotal Phase 3 PEACE trial (NCT03921541).

[0230] [Example]

[20] [:] [3] [Clinical trial design]

[0231] A randomized, double-blind, placebo-controlled Phase 3 study of the efficacy and safety of Co-rhARG1-PEG produced by the methods of the present invention is being conducted in children and adults with arginase 1 deficiency. This trial is currently being conducted in the following format: [ , P , ]egzilarginase [ , E , ]effect on [ , A , ]rginase 1 Deficiency [ , C , ]linical [ , E , ]ndpoints or PEACE (CAEB1102-300A; NCT03921541).

[0232] The study design for this Phase 3 trial is shown in the figure below:

[0233] Key inclusion criteria a. Aged ≥ 2 years, diagnosed with ARG1-D, and with plasma arginine levels ≥ 250 μmol / L to allow statistical testing of the proportion of patients whose plasma arginine levels reached below the medical guideline value of 200 μmol / L. b. Ability to maintain a stable, consistent diet during the blind period c. Ability to maintain stable doses of ammonia scavengers, anti-epileptic therapy, and / or anticonvulsant medication during the blind period d. Be able to perform and successfully complete clinical evaluations and must have baseline impairments in one of the components of the secondary clinical response endpoint as shown in Table 16.

[0234] Key exclusion criteria a. An episode of hyperammonemia requiring hospitalization within 6 weeks prior to starting treatment b. Active infection within 3 weeks before receiving the first dose of PEGylated arginine c. Extreme motor impairment, defined as inability to perform the Gillette Functional Assessment Questionnaire (GFAQ) or a GFAQ score of 1 (complete inability to walk) d. Participated in previous interventional studies using PEGylated arginine or is currently participating in other clinical trials e. History of allergy to polyethylene glycol

[0235] Table 16: Definition of Baseline Impairments for Key Clinical Response Endpoints 2MWD = 2-minute walk distance; GMFM = Gross Motor Function Measure, Section D = Stand; Section E = Walk, Run, Jump *NIH Toolbox (US Department of Health and Human Services, Washington, DC, USA) Exercise Data Set (2-Minute Walk Endurance Test)

[0236] The primary endpoint of this Phase 3 trial was plasma arginine reduction (change from baseline in plasma arginine levels at Week 24, based on treatment-dependent changes from baseline in individual patients in the active and placebo groups).

[0237] Secondary endpoints include: a. Clinical Response Endpoints: Clinical responders were defined as patients who had improvement in at least one of the 2MWT, GMFM-D, or GMFM-E clinical response endpoints at Week 24, as defined in Table 17. b. Response rate for each individual component of the clinical response endpoint c. Other clinical outcome evaluations i. Functional Movement Scale: 5m, 50m, 500m ii. Gillette Functional Assessment Questionnaire (GFAQ) iii. Vineland Adaptive Behavior Scale - II d. Safety (including immunogenicity) assessment e. Proportion of patients with plasma arginine <200 μM and within the normal range (40 - 115 μM) f. Characterization of the Pharmacokinetics of Pegylated Arginase

[0238] Table 17: Clinical Response Endpoints Defining Clinical Responders

[0239] The total duration of the study is expected to be approximately 178 weeks per subject, including a long-term open-label extension phase (a 3-4-week screening period, a 24-week treatment period, followed by an open-label extension phase of up to 150 weeks). Subjects received weekly intravenous infusions (approximately 30 minutes) of Co-rhARG1-PEG or volume-adjusted placebo once a week. Co-rhARG1-PEG dose modifications were made by an unblinded pharmacist and / or physician according to a dosing algorithm based solely on plasma arginine levels. Following the first 8 weeks of the long-term blinded extension phase, subjects were given the option of subcutaneously administering Co-rhARG1-PEG, with investigator and sponsor approval. The initial subcutaneous mg / kg dose was the same as the intravenous dose.

[0240] Subjects assigned to Co-rhARG1-PEG started at dose level 2 (0.10 mg / kg) (see Table 18 below). Starting at Visit 5, dose modifications were made as needed based on plasma arginine values by an unblinded physician according to the following dosing algorithm. ● If the plasma arginine level is >150 μM, then this sample will be used to increase the dose by 2 dose levels (not to exceed 0.20 mg / kg) if the 2 doses prior to a single 168-hour sample were a) the same dose level expressed in mg / kg and b) consecutive (no missed doses). • If plasma arginine levels from two consecutive 168-hour samples (regardless of whether a dose was missed) are <50 μM, reduce the dose by one dose level (see Table 17), but not to below 0.05 mg / kg.

[0241] Table 18: Dose adjustment of Co-rhARG1-PEG [Dose Level] [ , (a) , ] [dose] 1 (minimum possible dose) 0.05 mg / kg 2 (starting dose) 0.10 mg / kg 3 0.15 mg / kg 4 (maximum possible dose) 0.20 mg / kg a) Pegylated arginine should be administered starting at Level 2 (0.10 mg / kg). If necessary, the dose may be increased by 2 dose levels. The dose may be decreased by 1 dose level.

[0242] Statistical considerations

[0243] The primary analysis will compare the mean reduction from baseline in plasma arginine levels after 24 weekly doses in patients treated with pegarginase versus placebo, based on the mean of four plasma arginine measurements after meeting strict prespecified criteria.

[0244] A sample size of 10 and 20 patients randomized to placebo and peg-arginine, respectively, achieved 98% power to demonstrate a difference in mean plasma arginine levels of 200 μM at a significance level of 0.05 using a two-sided Mann-Whitney-Wilcoxon test, assuming a commonly used SD of 120 μM.

[0245] Additionally, this number of subjects provided greater than 80% power to detect a statistically significant difference of 40% between the group proportions for the clinical response endpoint using Fisher's Exact Test at a significance level of 0.05.

[0246] [Example] [twenty one] [:Site-specific PEGylation analysis]

[0247] An exemplary site-specific PEGylation analysis is provided in FIG15 . Three batches of Co-rhARG1-PEG were analyzed. As can be seen, none of the three batches were PEGylated at sites K3, K149, K190, K195, K29, K265, or K283. Additionally, all three batches were PEGylated at sites K16, K32, K38, K40, K47, K67, K74, K82, L87, K88, K152, K154, K171, K312, and K321. Certain batches had low PEGylation frequencies at sites K222 and K223.

[0248] References throughout this specification to "one embodiment," "certain embodiments," "various embodiments," "one or more embodiments," or "an embodiment" mean that the particular features, structures, materials, or characteristics described in connection with that embodiment are included in at least one embodiment of the present disclosure. Therefore, the appearances of phrases such as "in one or more embodiments," "in certain embodiments," "in various embodiments," "in an embodiment," or "in an embodiment" in various places throughout this specification are not necessarily referring to the same embodiment of the present disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments.

[0249] Although the disclosure herein is described with reference to specific embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the disclosure. Those skilled in the art will appreciate that various modifications and variations can be made to the disclosure without departing from its spirit and scope. Therefore, the disclosure is intended to include such modifications and variations as come within the scope of the appended claims and their equivalents.

Claims

1. A composition comprising cobalt-substituted polyethylene glycol recombinant human arginase (Co-rhARG-PEG), the Co-rhARG-PEG being produced by a method for producing purified recombinant cobalt-substituted human arginase, wherein the recombinant human arginase (rhARG) comprises an amino acid sequence identical to that of SEQ ID NO: 1, the method comprising: a. culturing rhARG-producing *E. coli* cells in a bioreactor at 36°C and 38°C and at a pH between 7.0 and 7.4, with stirring and aeration; b. lysing the *E. coli* cells by high-pressure homogenization in 25 mM HEPES at a pH between 7.2 and 7.6 and at 15°C or below; c. removing cell debris from the lysate; d. Load the cell lysate onto a cation exchange column (CEX) and wash the column with 25 mM HEPES at pH 7.2–7.6; e. Elute the rhARG with a high-salt solution containing 25 mM HEPES and 0.1 M NaC at pH 7.2–7.6 at room temperature; f. Incubate the eluted rhARG with 10 mM CoCl2 at room temperature for 2–8 hours to form cobalt-substituted rhARG (Co-rhARG), and exchange the Co-rhARG into 50 mM Tris at pH 8.1–8.5; g. Apply the Co-rhARG to an anion exchange column (AEX) and collect the flow-through; h. Apply the flow-through to a third chromatography column; and i. Elute the rhARG with a high-salt solution containing 50 mM Tris and 250 mM NaC at pH 8.1–8.

5. The Co-rhARG was eluted from the third chromatography column using a high-salt NaCl solution (buffer for the third chromatography column); (i) the buffer for the third chromatography column was replaced with 20 mM sodium phosphate, 50 mM NaCl and 1.5% glycerol at pH 7.4 (buffer 1), and the protein concentration was adjusted to 5.0 mg / mL; (ii) buffer 1 was replaced with 0.1 M sodium phosphate at pH 8.1–8.5 (buffer 2), and the protein concentration was adjusted to 10.0 mg / mL; and j. Reacting the Co-rhARG with a PEGylation reactant to provide a PEGylated protein; wherein the PEG:Co-rhARG molar ratio is in the range of 7 molar / molar to 15 molar / molar.

2. The composition as claimed in claim 1, 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.

3. A composition comprising a recombinant human arginase (rhARG) protein, wherein the protein comprises an amino acid sequence consistent with SEQ ID NO: 1, wherein the protein is misculated with a non-natural metal cofactor cobalt to form a cobalt-substituted rhARG (Co-rhARG), and wherein the protein is covalently linked to polyethylene glycol (PEG) at one or more sites of K16, K32, K38, K40, K47, K67, K74, K82, K87, K88, K152, K154, K171, K222, K223, K312 and K321, wherein the PEG:Co-rhARG molar ratio is in the range of 7 moles / moles to 15 moles / moles.

4. The composition as claimed in any one of claims 1 to 3, wherein the recombinant human argininase (rhARG) comprises an amino acid substitution at a position selected from the group consisting of H100, D123, H125, D127, D231, D233, W121, D180, S229, C302 and E255.

5. The composition as claimed in claim 3, wherein the recombinant human argininase (rhARG) comprises at least one amino acid substitution selected from the group consisting of D180S, S229C, S229G, C302F, C302I, E255Q, D180E and S229A.

6. The composition as claimed in claim 3, wherein the recombinant human argininase (rhARG) comprises at least one amino acid substitution located at C302.

7. The composition as claimed in claim 3, wherein the recombinant human argininase (rhARG) comprises at least two amino acid substitutions.

8. The composition as claimed in claim 3, wherein the recombinant human arginase (rhARG) is a truncated arginase I protein.

9. The composition as claimed in claim 3, wherein the recombinant human argininase (rhARG) further comprises a foreign protein fragment.

10. The composition of claim 9, wherein the exogenous protein fragment comprises the Fc region of an immunoglobulin or a portion thereof.

11. The composition as claimed in claim 1, wherein the specific activity of the Co-rhARG-PEG is in the range of 400 U / mg to 700 U / mg.

12. The composition as claimed in any one of claims 1 to 3, wherein when the protein is analyzed in vitro, it exhibits arginine hydrolysis kcat / KM in the range of 200 mM⁻¹ s⁻¹ to 4,000 mM⁻¹ s⁻¹ at pH 7.

4.

13. The composition of claim 12, wherein when the protein is analyzed in vitro, it exhibits arginine hydrolysis kcat / KM in the range of 400 mM⁻¹ s⁻¹ to 2,500 mM⁻¹ s⁻¹ at pH 7.

4.

14. The composition as claimed in claim 1, wherein the free PEG concentration is less than or equal to 100 µg / mL.

15. The composition as claimed in any one of claims 1 to 3, wherein the total cobalt content of the composition is in the range of 9 µg / mL to 15 µg / mL.

16. The composition as claimed in any one of claims 1 to 3, 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% to 30%, peak 5 is in the range of 15% to 30%, peak 6 is in the range of 10% to 25%, peak 7 is less than 25%, peak 8 is less than 15%, and peak 9 is less than 8%.

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

18. A pharmaceutical composition comprising Co-rhARG-PEG as described in any one of claims 1 to 17 and a pharmaceutically acceptable carrier.

19. The pharmaceutical composition as claimed in claim 18, wherein the composition is formulated for intravenous or subcutaneous administration.

20. The pharmaceutical composition as claimed in claim 18 or 19, wherein the composition comprises potassium phosphate, sodium chloride and glycerin.

21. The pharmaceutical composition as claimed in claim 20, wherein the composition comprises 50 mM NaCl, 1 mM K2HPO4, 4 mM KH2PO4 and 1.5% w / v glycerol.

22. Use of a pharmaceutical composition as described in any one of claims 18 to 21 in the preparation of a medicament for treating arginase 1 deficiency.

23. The use as described in claim 22, wherein the pharmaceutical composition is administered intravenously.

24. The use as described in claim 22, wherein the pharmaceutical composition is administered subcutaneously.

25. The use as described in any one of claims 22 to 24, wherein the administration of the pharmaceutical composition is based on the weight of the unPEGylated enzyme, starting at a dose of 0.1 mg / kg.

26. The use as described in any of claims 22 to 24 further includes monitoring a patient’s plasma arginine levels.

27. The use as described in claim 25, wherein the dose is adjusted according to the following algorithm: a. If the plasma arginine level is >150 μM, then if the two doses prior to a single 168-hour sample are a) the same dose level in mg / kg and b) consecutive (without missed doses), the sample will be used to increase the dose by two dose levels in the table below (not exceeding 0.20 mg / kg); b. If the plasma arginine levels (regardless of whether a dose was missed) from two consecutive 168-hour samples are both <50 μM, then the dose will be reduced by one dose level in the table below, but not to below 0.05 mg / kg; (a) polyethylene glycol arginase administration begins at level 2 (0.10 mg / kg), and the dose is increased by two dose levels or decreased by one dose level as needed.

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