Methods for producing human recombinant arginase 1 and uses thereof

Abstract

To provide methods for producing recombinant arginase such as PEGylated cobalt-substituted recombinant human arginase 1, pharmaceutical compositions containing such recombinant arginase as well as therapeutic methods and methods of using such a recombinant arginase.SOLUTION: Disclosed is a method for producing a purified recombinant cobalt-substituted human arginase (rhARG) having a specific sequence, where the method comprises the steps of: culturing E. coli cells that produce rhARG in a bioreactor; applying the cell lysate of the E. coli cells to a cation exchange column; incubating the eluted rhARG with a cobalt salt to form cobalt-substituted rhARG (Co-rhARG); applying the Co-rhARG to an anion exchange column to collect a flow-through from the column; applying the flow-through to a third chromatographic column and eluting Co-rhARG from the third column.SELECTED DRAWING: Figure 15

Description

Technical Field

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

Background Art

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

[0003] Arginase 1 is mainly found in red blood cells (RBCs) and the liver. ARG1 is the only gene currently known in which mutations cause arginase 1 deficiency. Clinically, arginase 1 deficiency is characterized by a slow deterioration of the cerebral cortex and pyramidal tracts leading to progressive dementia, psychomotor retardation, spastic diplegia, seizures, and growth retardation. Left untreated, the disease progresses to severe spasticity, loss of walking, loss of bowel and bladder control, and severe intellectual disability. Patients with arginase 1 deficiency typically exhibit elevated blood arginine levels (3 - 4 times the upper limit of normal [ULN]), mild hyperammonemia, and a mild increase in urinary orotic acid. Most patients do not have detectable arginase 1 enzyme activity in RBCs (< 1% of normal).

[0004] Current treatment of arginase 1 deficiency focuses on maintaining plasma arginine concentration as close to normal levels as possible through lifelong dietary protein restriction. Protein intake is limited to the minimum necessary to maintain protein biosynthesis and growth. More than half of dietary protein is provided in the form of an essential amino acid mixture that does not contain arginine. Such dietary changes can lower plasma arginine levels in most patients, but this diet is unappetizing, expensive, and particularly difficult to maintain and manage in growing children.

[0005] The paucity of treatment options for patients with arginase 1 deficiency highlights an important unmet need for treatments that lower arginine levels within the normal range and promote the maintenance of normal arginine levels throughout life. The development of such therapies may help in the attempt to minimize exposure to the neurotoxic effects of arginine and its metabolites and provide the possibility of normal neurocognitive development in these patients.

[0006] In addition to the treatment of arginase 1 deficiency or hyperargininemia, arginase produced by these methods can be used in the treatment of other diseases. Arginase 1 has been used in clinical trials investigating its use in cancer treatment and is used in combination with immuno-oncology drugs such as pembrolizumab. SUMMARY OF THE INVENTION

[0007] Production of Arginase One aspect of the invention relates to a method for producing and / or purifying a recombinant human arginase protein. In one or more embodiments, the recombinant human arginase protein is recombinant human arginase 1 (rhARG1) (shown in SEQ ID NO: 1, FIG. 1(a)). In other embodiments, the recombinant human arginase protein is recombinant human arginase 2 (rhARG2) (see SEQ ID NO: 3, FIG. 1(c)). Although specifically referred to herein as rhARG1, the methods, formulations, and uses described herein can also be applied to rhARG2.

[0008] The human arginase 1 and 2 proteins used in the method of the present invention have two Mn2+ sites, and either or both sites can be substituted to generate a modified arginase 1 or 2 protein with a non-native metal cofactor. In some embodiments, the protein exhibits a kcat / KM greater than 200 mM-1 s-1 at pH 7.4. In certain embodiments, the protein exhibits a kcat / KM in the range of about 200 mM-1 s-1 to about 4,000 mM-1 s-1 at pH 7.4. In another embodiment, the protein exhibits a kcat / KM in the range of about 400 mM-1 s-1 to about 2,500 mM-1 s-1 at 37 °C and pH 7.4. In certain embodiments, the present invention contemplates a protein comprising the amino acid sequence of human arginase 1 or 2 and a non-native metal cofactor, wherein the protein exhibits a kcat / KM greater than 400 mM-1 s-1 at 37 °C and pH 7.4. Exemplary kcat / KM values include about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 800, about 900, about 1,000, about 1,100, about 1,200, about 1,500, about 2,000, about 3,000, about 3,500 and about 4,000 mM-1 s-1 at 37 °C and pH 7.4.

[0009] In one or more embodiments, a method for producing a recombinant cobalt-substituted human arginase protein (Co-rhARG) is provided. In one or more embodiments, the method includes: fermenting a strain of E. coli cells that express rhARG1, providing a Co-arginase 1 intermediate (Co-rhARG1) using cobalt instead of manganese in rhARG1, purifying the Co-arginase 1 intermediate and PEGylating the Co-arginase 1 intermediate to form a prodrug (Co-rhARG1-PEG). In one or more embodiments, Co-rhARG1-PEG comprises pegzilarginase.

[0010] In one or more embodiments, the method comprises several steps: culturing E. coli cells in a bioreactor that produces recombinant human arginase (rhARG), lysing the E. coli cells, removing cell debris from the lysate, loading the cell lysate onto a cation exchange column, eluting the recombinant human arginase protein (rhARG) with a high salt concentration solution, incubating the eluted recombinant human arginase protein (rhARG) with a cobalt salt to form a cobalt-substituted recombinant human arginase protein (Co-rhARG), applying the cobalt-substituted recombinant human arginase protein (Co-rhARG) to an anion exchange column and collecting the flow-through, adding the flow-through to a third chromatography column, and eluting the cobalt-substituted recombinant human arginase protein (Co-rhARG) from the third chromatography column at a high salt concentration.

[0011] In one or more embodiments, the method for producing a recombinant cobalt-substituted human arginase protein (Co-rhARG) comprises loading a maximum of 60 grams of recombinant human arginase protein (rhARG) per liter of cation exchange resin onto a cation exchange column.

[0012] In one or more embodiments, the method for producing a recombinant cobalt-substituted human arginase protein (Co-rhARG) comprises eluting the recombinant human arginase protein (rhARG) from a cation exchange column using a high salt concentration solution with a maximum salt concentration of about 0.5 M. In some embodiments, the recombinant human arginase protein (rhARG) is eluted from the cation exchange column using a high salt concentration solution with a concentration of 0.1 M. In some embodiments, the recombinant human arginase protein (rhARG) is eluted from the cation exchange column using a gradient of about 0.0 to about 0.5 M salt concentration. In some embodiments, the recombinant human arginase protein (rhARG) is eluted from the cation exchange column using a gradient of about 0.0 to about 0.2 M salt concentration.

[0013] In one or more embodiments, a method for producing a recombinant cobalt-substituted human arginase protein (Co-rhARG) includes incubating a recombinant human arginase protein (rhARG) eluted from a cation exchange column with a cobalt salt containing Co2+. In some embodiments, the cobalt salt includes CoCl2.

[0014] In one or more embodiments, a method for producing a recombinant cobalt-substituted human arginase protein (Co-rhARG) includes adding a recombinant cobalt-substituted human arginase protein (Co-rhARG) flow-through to a third chromatography column that includes a multimodal chromatography (MMC) column.

[0015] In one or more embodiments, a method of producing a recombinant cobalt-substituted human arginase protein (Co-rhARG) comprises reacting a recombinant cobalt-substituted human arginase protein (Co-rhARG) or a recombinant cobalt-substituted human arginase protein (Co-rhARG) with a PEGylation reactant to provide a PEGylated protein. In some embodiments, the PEGylated protein comprises one or more of the PEGylated amino acid residues at K16, K32, K38, K40, K47, K67, K74, K82, L87, K88, K152, K154, K171, K222, K223, K312, and K321. In some embodiments, the PEGylated protein comprises one or more of about 15% to about 60% of K16, about 35% to about 80% of K32, about 20% to about 85% of K38, about 10% to about 60% of K40, about 10% to about 60% of K47, about 40% to about 90% of K67, about 30% to about 95% of K74, about 30% to about 98% of K82, about 15% to about 65% of K87, about 25% to about 70% of K88, about 25% to about 85% of K152, about 15% to about 65% of K154, about 20% to about 75% of K171, 0% to about 30% of K222, 0% to about 35% of K223, 0% to about 45% of K312, and 0% to about 45% of K321 being PEGylated. In some embodiments, the PEGylated protein comprises PEGylated amino acid residues at least at K16, K32, K38, K40, K47, K67, K74, K82, L87, K88, K152, K154, K171, K312, and K321. In some embodiments, the PEGylated protein does not have PEGylated amino acid residues at K3, K149, K190, K195, K29, K265, and K283.

[0016] One or more embodiments of Co-rhARG1-PEG relate to a cobalt-substituted PEGylated human recombinant arginase 1 enzyme expressed in E. coli formulated for intravenous (IV) or subcutaneous (SC) administration. Replacing native manganese (Mn2+) with cobalt (Co2+) at the active site of arginase 1 improves stability and catalytic activity at physiological pH. PEGylation also extends the circulation half-life (t1 / 2) of recombinant arginase 1.

[0017] In various embodiments, the method includes culturing E. coli cells in a bioreactor to produce recombinant human arginase 1, lysing the E. coli cells, and purifying the recombinant human arginase 1 (see FIGS. 2 and 3). Purification of the Co-arginase 1 intermediate can be performed by a purification procedure that includes one or more of the following steps: cell disruption by high-pressure homogenization, clarification of the homogenate, SP Sepharose FF cation-exchange capture chromatography, cobalt exchange, ultrafiltration / diafiltration, Q Sepharose FF anion-exchange flow-through chromatography, Capto MMC multimodal chromatography, and ultrafiltration / diafiltration. The purified Co-arginase 1 intermediate is either processed to form a PEGylated drug substance or frozen and stored for later conversion to the drug substance.

[0018] In a preferred embodiment of this method, an E. coli lysate containing rhARG1 is loaded onto a cation-exchange (CEX) chromatography column (also referred to as "Column 1") to capture rhARG1, which is then eluted with a high-salt concentration solution to provide a first protein product ("the first protein product").

[0019] In one or more embodiments, the method further includes 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 ("the second protein product"). In another aspect of this method, the method further includes loading the second protein product onto a multimodal chromatography (MMC) column that captures arginase 1 and is then eluted to provide a third protein product ("the third protein product"). In some embodiments, this third chromatography column (also referred to as "Column 3") can be a size-exclusion chromatography (SEC) column.

[0020] Various embodiments involve modifying the native manganese coenzyme of arginase for a cobalt coenzyme. Cobalt substitution (also referred to as cobalt loading) can be performed at any stage of the manufacturing process. For example, cobalt loading of arginase 1 can be performed at any of E. Coli lysate, the first protein product, the second protein product, the third protein product, or any step of PEGylated arginase 1. In other embodiments, cobalt loading can be performed on arginase 1 eluted from column 1, arginase 1 eluted from column 2, or arginase 1 eluted from column 3. Cobalt loading can be performed on arginase 1 eluted from a CEX column, arginase 1 eluted from an AEX column, arginase 1 eluted from an MMC column, or arginase 1 eluted from an SEC column.

[0021] Cobalt loading of arginase 1 can be performed at various temperatures using various solutions containing cobalt. In one or more embodiments, the cobalt salt contains Co2+ such as CoCl2. In a preferred embodiment, cobalt loading of arginase 1 is performed using CoCl2 at room temperature or near room temperature, such as about 15 to about 25 °C or about 20 to about 25 °C. The cobalt loading rate can be manipulated by raising or lowering the reaction temperature. Cobalt loading can also be performed at various pH values.

[0022] One aspect of the present disclosure relates to varying the conditions associated with CEX chromatography (column 1). The amount of protein loaded onto column 1 can be increased or decreased to select for various arginase 1 charge variants. Manipulating the loading factor can cause a shift to a more desirable CEX charge species profile. A loading factor of up to about 60 g / L (amount of protein in grams / volume of CEX column resin in liters) can produce arginase 1 with a 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.

[0023] In a preferred embodiment, arginase 1 is first captured on column 1, subsequently purified continuously on column 2, and then captured on column 3. In another embodiment, an E. coli lysate can be loaded onto an AEX column (e.g., column 2), and the flow-through can be applied to a CEX column to capture arginase 1. In another embodiment, the cobalt loading of arginase 1 can occur after the PEGylation reaction. Also, other chromatography columns such as SEC columns can be used to replace the MMC column.

[0024] One aspect of the present invention relates to a method for producing a recombinant cobalt-substituted human arginase protein (Co-rhARG). In one or more embodiments, the recombinant human arginase protein (rhARG) comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1. The method consists of several steps: culturing E. coli cells in a bioreactor that produces recombinant human arginase (rhARG), lysing the E. coli cells, removing cell debris from the lysate, loading the cell lysate onto a cation exchange column, eluting the recombinant human arginase protein (rhARG) with a high salt concentration solution, incubating the eluted recombinant human arginase protein (rhARG) with a cobalt salt to form a cobalt-substituted recombinant human arginase protein (Co-rhARG), applying the cobalt-substituted recombinant human arginase protein (Co-rhARG) to an anion exchange column and collecting the flow-through, adding the flow-through to a third chromatography column, eluting the cobalt-substituted recombinant human arginase protein (Co-rhARG) from the MMC column with a high salt concentration solution, reacting with a molar excess of methoxy PEG succinimidyl carboxymethyl ester, and removing the excess PEG.

[0025] Recombinant human arginase 1, pharmaceutical compositions and formulations Another aspect of the present invention relates to rhARG1, Co-rhARG1 and / or Co-rhARG1-PEG produced by the methods described herein, or compositions containing the same.

[0026] In one or more embodiments, the protein is covalently conjugated to polyethylene glycol at one or more of K16, K32, K38, K40, K47, K67, K74, K82, L87, K88, K152, K154, K171, K222, K223, K312, and K321.

[0027] Another aspect of the invention relates to a composition comprising a recombinant human arginase (rhARG) protein, the protein comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 1, the protein being a complex with a non-natural metal cofactor, the non-natural metal cofactor being cobalt, and the protein being covalently conjugated to polyethylene glycol at one or more of K16, K32, K38, K40, K47, K67, K74, K82, L87, K88, K152, K154, K171, K222, K223, K312, and K321.

[0028] In one or more embodiments, the protein 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.

[0029] In one or more embodiments, the protein comprises at least one amino acid substitution selected from the group consisting of: D180S, S229C, S229G, C302F, C302I, E255Q, D180E, and S229A.

[0030] In one or more embodiments, at least one amino acid substitution is at C302.

[0031] In one or more embodiments, the protein comprises at least two amino acid substitutions.

[0032] In one or more embodiments, the protein is a truncated arginase I protein.

[0033] In one or more embodiments, the protein further comprises an exogenous protein fragment.

[0034] In one or more embodiments, the exogenous protein fragment comprises the Fc region of an immunoglobulin or a portion of the Fc region of an immunoglobulin.

[0035] In one or more embodiments, the specific activity of Co-rhARG-PEG is in the range of about 400 U / mg to about 700 U / mg.

[0036] In one or more embodiments, when assayed in vitro, the protein exhibits a kcat / Km for arginine hydrolysis in the range of about 200 mM−1s−1 to about 4,000 mM−1s−1 at pH 7.4.

[0037] In one or more embodiments, when assayed in vitro, the protein exhibits a kcat / KM for arginine hydrolysis in the range of about 400 mM−1 s−1 to about 2,500 mM−1 s−1 at pH 7.4.

[0038] In one or more embodiments, the molar ratio of PEG:Co-rhARG is in the range of about 7 moles / mole to about 15 moles / mole.

[0039] In one or more embodiments, the free PEG concentration is 100 μg / mL or less.

[0040] In one or more embodiments, the total cobalt content of the composition is in the range of about 9 μg / mL to about 15 μg / mL.

[0041] In one or more embodiments, when loaded onto imaging capillary isoelectric focusing (iCIEF), the composition generates at least nine peaks, where peak 1 is less than 20%, peak 2 is less than 30%, peaks 3 + 4 are in the range of 10 - 30%, peak 5 is in the range of 15 - 30%, peak 6 is in the range of 10 - 25%, peak 7 is less than 25%, peak 8 is less than 15%, and peak 9 is less than 8%.

[0042] In one or more embodiments, the composition generates at least nine peaks when loaded onto icIEF, with peak 1 in the range of 5-7%, peak 2 in the range of 8-11%, peak 3+4 in the range of 16-20%, peak 5 in the range of 21-24%, peak 6 in the range of 21-22%, peak 7 in the range of 14-15%, peak 8 in the range of 5-8%, and peak 9 in the range of 2-3%.

[0043] Another aspect of the invention relates to a pharmaceutical composition 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 about 50 mM NaCl, about 1 mM K2HPO4, about 4 mM KH2PO4, and about 1.5% w / v glycerol.

[0044] Treatment method for arginase 1 deficiency Another aspect of the invention relates to the administration of recombinant human arginase 1 such as Co-rhARG1-PEG. Such administration can be by any suitable method including IV or SC administration. In one or more embodiments of this aspect, the dose of Co-rhARG1-PEG is determined by a specific algorithm: In one or more embodiments of this algorithm, the patient starts treatment at 0.10 mg / kg. Plasma arginine levels are monitored. If the plasma arginine level is >150 μM, the dose is increased to 0.20 mg / kg. If the plasma arginine level is <50 μM, the dose is decreased to 0.05 mg / kg. Otherwise, the patient maintains a dose of 0.10 mg / kg.

[0045] In one or more embodiments of this algorithm, the dose changes are as follows: · If the plasma arginine level is >150 μM, the two doses prior to this sample are at the same dose level of a) mg / kg, b) If continuous (no missed dosing), increase the dose by only 2 dose levels in the table below (not exceeding 0.20 mg / kg) using a single 168-hour sample. · If plasma arginine levels from both of 2 consecutive 168-hour samples (even in case of missed dosing) are both < 50 μM, decrease the dose by only 1 dose level in the table below, such that it does not fall below 0.05 mg / kg.

Table 1

Brief Description of the Drawings

[0046] Further features of the present invention will become apparent from the following written description and the accompanying drawings.

[0047]

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Figure 15

[0048] Recombinant Human Arginase 1 Human arginase 1, identified as hArg1, is a binuclear manganese metalloenzyme that catalyzes the hydrolysis of L-arginine (L-Arg) to produce L-ornithine and urea. Arginase 1 is a trimer of three non-covalently linked identical monomeric units. Monomeric arginase 1 is enzymatically active but has low stability. Substituting native manganese (Mn2+) with cobalt (Co2+) at the active site of arginase 1 improves the catalytic activity at physiological pH. The method for producing the cobalt-substituted arginase 1 enzyme described herein provides a high-purity and highly active enzyme. This method can also provide Co-arginase 1 (Co-rhARG1) as an isolated intermediate in the manufacture of a drug substance. In one or more embodiments, the drug substance is PEGylated Co-arginase 1 (Co-rhARG1-PEG). PEGylation of Co-arginase 1 significantly extends the circulation half-life. Again, although rhARG1 is specifically referred to herein, the methods, formulations, and uses described herein can also be applied to rhARG2.

[0049] As used herein, the term "rhARG1" refers to a recombinant human arginase 1 enzyme, such as a recombinant enzyme having at least 98% sequence identity to SEQ ID NO: 1.

[0050] As used herein, terms such as "Co-rhARG1", "Co-arginase 1 intermediate" refer to rhARG1 in which at least a portion of the native manganese cofactor has been replaced with cobalt. In one or more embodiments, Co-rhARG1 is an isolable intermediate in the production and / or purification process of Co-rhARG1-PEG.

[0051] As used herein, terms such as "Co-rhARG1-PEG", "PEGylated Co-arginase 1" refer to Co-rhARG1 having one or more PEG units covalently attached to the enzyme, such as the free amine(s) of the N-terminal amino acid and / or one or more lysine residues.

[0052] The amount of Co-rhARG1-PEG drug substance can be expressed as the amount of the non-PEGylated enzyme assembly. In one embodiment of this method, each mg (enzyme basis) of Co-rhARG1-PEG drug substance also contains approximately 1-2 mg of PEG, such as about 1 mg of PEG, in 1.4 mg.

[0053] 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 P05089). Overlapping oligonucleotides were used in the PCR reaction to generate arginase 1 DNA with codons optimized for expression in E. coli (Figure 1(b)). The 321-amino acid E. coli-expressed monomer of arginase 1 lacks the N-terminal methionine found in the native human arginase 1 monomer. 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 have disulfide bonds.

Table 2

[0054] In one or more embodiments, the calculated molecular weight of monomer Co-rhARG1-PEG is about 75-115 kDa. In one or more embodiments, the calculated molecular weight of homotrimer Co-rhARG1-PEG is about 224-344 kDa. In one or more embodiments, the average number of PEGs is about 8 to about 25 moles of PEG / mole of Co-arginase 1 monomer, for example, about 8 to about 16 moles of PEG / mole of Co-arginase 1 monomer. Exemplary amounts of PEG include about 8, about 9, about 10, about 11, about 12, about 13, about 14, and about 16 moles of PEG / mole of Co-arginase 1 monomer. The average molecular weight of each PEG is 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 certain embodiments, the average MW of PEG is about 5,000 daltons.

[0055] In one or more embodiments, Co-rhARG1-PEG comprises pegzilarginase. Pegzilarginase has the following two chemical names: a. Poly(oxy-1,2-ethanediyl), α-(carboxymethyl)-ω-methoxy-, amide with arginase 1 [cobalt cofactor] (synthetic human) (1:10), trimer b. Des-Met1-arginase-1 (liver-type arginase, EC 3.5.3.1) (Homo sapiens), non-covalently bound homotrimer, produced in Escherichia coli, where manganese is replaced by cobalt and the average of 10 primary amines (of the N-terminal serine and N6-lysine) are amidated with [methoxypoly(ethyleneoxy)]acetyl. The molecular formula of pegzilarginase is C1554H2492N416O453S6 [C3H4O2(C2H4O)n]a monomer. The average molecular weight of pegzilarginase is 284 kDa as a trimer. The CAS registration number of pegzilarginase is 1659310-95-8.

[0056] The possible sites of PEGylation of pegzilarginase are shown below. [Chemical formula]

[0057] Generally, the PEGylation reaction is carried out with Co-rhARG1. In some embodiments, the PEGylation reaction can be carried out with respect to rhARG1. In one or more embodiments, the amounts of reactants, time, temperature, and handling of solutions and reactants (such as mixing, addition rate, handling of PEG, etc.) are important for producing a consistent PEGylated product. Generally, the PEGylation reaction with Co-rhARG1 is carried out in a reaction buffer at pH 8.4. In one or more embodiments, Co-rhARG1 is PEGylated with 0.1 M sodium phosphate buffer at pH 8.4. In one or more embodiments, the PEGylation reaction includes a reactant ratio in the range of PEG (g) to Co-rhARG1 (g) of 4:1 to 1:1. In one or more embodiments, the PEGylation reaction includes a reactant ratio in which PEG (g) to Co-rhARG1 (g) is about 2.77:1. In one or more embodiments, the PEGylation reaction is carried out by misting PEG and Co-rhARG1 for about 5 minutes to about 300 minutes, about 10 minutes to about 300 minutes, about 20 minutes to about 300 minutes, about 30 minutes to about 300 minutes, about 5 minutes to about 280 minutes, about 10 minutes to about 280 minutes, about 20 minutes to about 280 minutes, about 30 minutes to about 280 minutes, about 5 minutes to about 260 minutes, about 10 minutes to about 260 minutes, about 20 minutes to about 260 minutes, about 30 minutes to about 260 minutes, about 5 minutes to about 240 minutes, about 10 minutes to about 240 minutes, about 20 minutes to about 240 minutes, about 30 minutes to about 240 minutes. In one or more embodiments, the PEGylation reaction is stopped by removing excess PEG and lowering the pH of the reaction buffer. In some embodiments, the excess PEG is removed by filtration techniques. In one or more embodiments, the pH is lowered by exchanging the reaction buffer with a storage buffer. In some embodiments, the storage buffer is composed of 5 mM potassium phosphate, 50 mM NaCl, 1.5% w / v glycerol, and pH 7.4.

[0058] In one or more embodiments, Co-rhARG1-PEG is PEGylated at one or more of the K16, K32, K38, K40, K47, K67, K74, K82, L87, K88, K152, K154, K171, K222, K223, K312, and K321 amino acid residues. In some embodiments, Co-rhARG1-PEG is PEGylated at at least K16, K32, K38, K40, K47, K67, K74, K82, L87, K88, K152, K154, K171, K312, and K321 amino acid residues. In some embodiments, Co-rhARG1-PEG is PEGylated at the K222 and / or K223 amino acid residues. In some embodiments, the Co-rhARG1-PEG is not PEGylated at the K222 and / or K223 amino acid residues. In some embodiments, Co-rhARG1-PEG is not PEGylated at one or more of the K3, K149, K190, K195, K29, K265, and K283 amino acid residues. In some embodiments, Co-rhARG1-PEG is not PEGylated at the K3, K149, K190, K195, K29, K265, and K283 amino acid residues.

[0059] In one or more embodiments of Co-rhARG1-PEG, K16 is PEGylated in the range of about 15% to about 60%. In one or more embodiments of Co-rhARG1-PEG, K32 is PEGylated in the range of about 35% to about 80%. In one or more embodiments of Co-rhARG1-PEG, K38 is PEGylated in the range of about 20% to about 85%. In one or more embodiments of Co-rhARG1-PEG, K40 is PEGylated in the range of about 10% to about 60%. In one or more embodiments of Co-rhARG1-PEG, K47 is PEGylated in the range of about 10% to about 60%. In one or more embodiments of Co-rhARG1-PEG, K67 is PEGylated in the range of about 40% to about 90%. In one or more embodiments of Co-rhARG1-PEG, K74 is PEGylated in the range of about 30% to about 95%. In one or more embodiments of Co-rhARG1-PEG, K82 is PEGylated in the range of about 30% to about 98%. In one or more embodiments of Co-rhARG1-PEG, K87 is PEGylated in the range of about 15% to about 65%. In one or more embodiments of Co-rhARG1-PEG, K88 is PEGylated in the range of about 25% to about 70%. In one or more embodiments of Co-rhARG1-PEG, K152 is PEGylated in the range of about 25% to about 85%. In one or more embodiments of Co-rhARG1-PEG, K154 is PEGylated in the range of about 15% to about 65%. In one or more embodiments of Co-rhARG1-PEG, K171 is PEGylated in the range of about 20% to about 75%. In one or more embodiments of Co-rhARG1-PEG, K222 is PEGylated in the range of 0% to about 30%. In one or more embodiments of Co-rhARG1-PEG, K223 is PEGylated in the range of 0% to about 35%. In one or more embodiments of Co-rhARG1-PEG, K312 is PEGylated in the range of 0% to about 45%. In one or more embodiments of Co-rhARG1-PEG, K321 is PEGylated in the range of 0% to about 45%.

[0060] The PEG-protein molar ratio is a characteristic indicating the degree of PEGylation. In one or more embodiments, about 1 to about 20 moles of PEG were used to PEGylate 1 mole of Co-rhARG1. Exemplary ranges of the PEG:Co-rhARG1 molar ratio include 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, and 20:1. In some embodiments, the PEG:Co-rhARG molar ratio is in the range of about 7 moles / mole to about 15 moles / mole.

[0061] Free PEG is measured to demonstrate PEG clearance and stability. In some embodiments, the free PEG concentration (μg) in PEGylated Co-rhARG1 (mL) is 500 μg / mL or less, 400 μg / mL or less, 300 μg / mL or less, 200 μg / mL or less, 100 μg / mL or less, and 50 μg / mL or less.

[0062] Human arginase 1 catalyzes the fifth and final step of the urea cycle, the conversion of L-arginine to L-ornithine and urea. Co-rhARG1-PEG, the PEGylated prodrug, catalyzes the same reaction. The assay for evaluating enzyme activity measures the conversion of L-arginine to L-ornithine during a fixed reaction time at pH 7.4 and 37°C. The amount of product conversion is converted to the reaction rate, and Km and kcat are determined by fitting to the Michaelis-Menten equation.

Number

[0063] Vmax is the maximum reaction rate achieved at the saturated substrate concentration, and Km is the Michaelis-Menten binding constant for measuring the substrate concentration that gives a rate of half of Vmax. The enzyme turnover number, kcat, is calculated by Vmax / [E].

[0064] Specific activity is determined by dividing the reaction rate at 2 mM arginine, expressed in μmol / min, by the enzyme concentration expressed in mg.

[0065] The values of KM and kcat of Co-rhARG1-PEG prodrug measured in the enzyme activity assay are typically in the ranges of 0.15 - 0.22 mM and about 200 - 300 / sec, respectively. When the Co-arginase 1 intermediate is PEGylated to form the prodrug, the enzyme activity does not change significantly compared to the non-PEGylated intermediate. However, PEGylation significantly extends the circulation half-life of the Co-rhARG1-PEG formulation compared to the Co-arginase 1 intermediate.

[0066] In one or more embodiments, the protein (e.g., Co-rhARG1 or Co-rhARG1-PEG) exhibits a kcat / KM greater than 200 mM−1s−1 at pH 7.4. In certain embodiments, the protein exhibits a kcat / KM in the range of about 200 mM−1s−1 to about 4,000 mM−1s−1 at pH 7.4. In another embodiment, the protein exhibits a kcat / KM in the range of about 400 mM−1s−1 to about 2500 mM−1s−1 at 37 °C and pH 7.4. In certain embodiments, the present invention contemplates a protein comprising the amino acid sequence of human arginase 1 and a non-native metal cofactor, which protein exhibits a kcat / KM greater than 400 mM−1s−1 at 37 °C and pH 7.4. Exemplary kcat / KM values are at 37 °C and pH 7.4 and are 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 between these values is included.

[0067] Specific activity is an indicator of the potency of a protein (e.g., Co-rhARG1 or Co-rhARG1-PEG). In one or more embodiments, the specific activity of Co-rhARG-PEG is in the range of about 200 U / mg to about 1000 U / mg. Exemplary ranges of specific activity include about 200 U / mg to about 1000 U / mg, about 300 U / mg to about 1000 U / mg, about 400 U / mg to about 1000 U / mg, about 200 U / mg to about 900 U / mg, about 300 U / mg to about 900 U / mg, about 400 U / mg to about 900 U / mg, about 200 U / mg to about 800 U / mg, about 300 U / mg to about 800 U / mg, about 400 U / mg to about 800 U / mg, about 200 U / mg to about 700 U / mg, about 300 U / mg to about 700 U / mg, and about 400 U / mg to about 700 U / mg.

[0068] In one or more embodiments, rhARG1, Co-rhARG1, or Co-rhARG1-PEG can 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 can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more deletions, substitutions, and / or insertions with respect to the amino acid sequence set forth by SEQ ID NO: 1. Various alignment algorithms and / or programs can be used to calculate the identity between two sequences, including those available on the website of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0069] In one or more embodiments, rhARG1, Co-rhARG1, or Co-rhARG1-PEG has at least one amino acid substitution at a position selected from H100, D123, H125, D127, D231, D233, D180, S229, and C302. In some embodiments, rhARG1, Co-rhARG1, or Co-rhARG1-PEG comprises at least one amino acid substitution selected from the group consisting of: D180S, S229C, S229G, C302F, C302I, E255Q, D180E, and S229A. In one or more embodiments, rhARG1, Co-rhARG1, or Co-rhARG1-PEG comprises an amino acid substitution at least at C302.

[0070] Using the methods described herein, almost all of the manganese cofactors of arginase 1 can be replaced with cobalt. The change to the cobalt cofactor causes the Km of arginine to change from 2.8 mM to about 0.18 mM at pH 7.4. In one or more embodiments, Co-rhARG1-PEG contains about 0.1 to about 2 μg of 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 of Co / mg protein.

[0071] Free cobalt is a measure for demonstrating the clearance and stability of cobalt. In some embodiments, free cobalt is 0.10 μg / mL or less, 0.09 μg / mL or less, 0.08 μg / mL or less, 0.07 μg / mL or less, 0.06 μg / mL or less, 0.05 μg / mL or less, and 0.04 μg / mL or less.

[0072] Total cobalt affects the potency of the protein and serves as an indicator of bound cobalt because the amount of free cobalt is relatively low. In some embodiments, the total cobalt concentration ranges from about 5 μg / mL to about 20 μg / mL, from about 6 μg / mL to about 20 μg / mL, from about 7 μg / mL to about 20 μg / mL, from about 8 μg / mL to about 20 μg / mL, from about 9 μg / mL to about 20 μg / mL, from about 5 μg / mL to about 19 μg / mL, from about 6 μg / mL to about 19 μg / mL, from about 7 μg / mL to about 19 μg / mL, from about 8 μg / mL to about 19 μg / mL, from about 9 μg / mL to about 19 μg / mL, from about 5 μg / mL to about 18 μg / mL, from about 6 μg / mL to about 18 μg / mL, from about 7 μg / mL to about 18 μg / mL, from about 8 μg / mL to about 18 μg / mL, from about 9 μg / mL to about 18 μg / mL, from about 5 μg / mL to about 17 μg / mL, from about 6 μg / mL to about 17 μg / mL, from about 7 μg / mL to about 17 μg / mL, from about 8 μg / mL to about 17 μg / mL, from about 9 μg / mL to about 17 μg / mL, from about 5 μg / mL to about 16 μg / mL, from about 6 μg / mL to about 16 μg / mL, from about 7 μg / mL to about 16 μg / mL, from about 8 μg / mL to about 16 μg / mL, from about 9 μg / mL to about 16 μg / mL, from about 5 μg / mL to about 15 μg / mL, from about 6 μg / mL to about 15 μg / mL, from about 7 μg / mL to about 15 μg / mL, from about 8 μg / mL to about 15 μg / mL, and from about 9 μg / mL to about 15 μg / mL.

[0073] In various embodiments, Co-rhARG1-PEG contains less than about 1 μg of 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 of Mn / mg protein. In certain embodiments, the Co-rhARG1-PEG drug substance contains about 2 μg of Co / mg protein and about 0.05 μg of Mn / mg protein.

[0074] In various embodiments, Co-rhARG1-PEG contains less than about 1 μg of 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 of Fe / mg protein.

[0075] Production and purification of rhARG1, Co-rhARG1 and PEGrhARG1 An overview of exemplary upstream and downstream production methods can be seen in Figures 2 and 3.

[0076] Scale-up in shake flasks The purpose of scale-up / fermentation in shake flasks is to generate inoculum material for seeding the production fermenter. Scale-up in shake flasks creates a cell population for inoculating the production reactor and an extra cell population for analytical purposes. A representative overview of the arginase 1 fermentation process is shown in Figure 2.

[0077] An aliquot of the inoculation medium is introduced into one 500 mL flask (primary flask) and six 3 L disposable flasks (secondary flasks). The flasks are autoclaved and post-sterilization additives are transferred to each flask. Prior to inoculation, the primary medium is preheated to a processing temperature of 37 °C. Prior to secondary inoculation, the secondary flasks are preheated to a processing temperature of 37 °C.

[0078] Remove one vial from the refrigeration and thaw it out of the E. coli working cell bank (WCB) expressing arginase 1. Aseptically add the target amount (about 1.1 mL) of the thawed cells to the primary flask and incubate the flask with stirring at 37°C. Starting from several hours after inoculation, take samples from the flask every hour and track cell growth by the optical density at 600 nm (OD600). When the 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 with stirring at 37°C. Take samples from one secondary flask starting from 4 hours after inoculation every hour, and increase the sampling frequency to every 30 minutes when the OD600 reaches 1.5 or more. When the measured value reaches the specified density of OD600 of ≥2.0, sample the remaining secondary flasks. 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 fermenter. A typical overview of the fermentation process of arginase 1 is shown in Figure 2.

[0079] Production fermentation The purpose of production fermentation is to scale up the shake flask culture and induce the production of arginase 1. Production fermentation can generate a large amount of arginase 1. Following the scale-up stage in a shake flask for constructing a cell population, the fermentation process produces arginase 1 (in E. coli) as a soluble protein. In one embodiment, a 1500 L fermenter contains an initial batch medium containing pre-inoculation sterilized additives. After inoculation, the inputs to the fermenter include a nutrient feed, an antifoam solution, and the addition of an acid or base to maintain the pH of the culture broth. A secondary vessel holds the nutrient feed medium. An automatic control strategy maintains important parameters for consistent cell growth, such as dissolved oxygen, sparge rate, agitation rate, pH, pressure, temperature, etc. The expression of arginase 1 is induced by the addition of IPTG (isopropyl betta-D-1-thiogalactophranoside), and harvesting is performed approximately 18 hours later. The performance of the fermenter is evaluated by monitoring the cell density, percent solids, and the proportion of soluble arginase 1 at the end of production.

[0080] In a preferred embodiment, the fermentation medium is prepared directly in the production fermenter. Before in-situ sterilization (SIP), purified water is added to the fermentation medium until it reaches the required weight. The post-sterilization additives of kanamycin, glucose, and potassium phosphate are filter-sterilized into the production fermenter after the medium has cooled. If necessary, a 0.2 μm sterilizing filter is used to bring the sterilized medium to the specified pre-inoculation weight with purified water. The fermentation medium is titrated with a base (ammonium hydroxide) to a controlled pH value.

[0081] The 37°C production fermenter is aseptically inoculated using inoculum pooled via pressure-assisted transfer. Fermentation broth samples are collected periodically from inoculation until fermentation cools down and measured for OD600 analysis. Glucose samples are taken starting 3 hours after inoculation and at regular intervals with increased frequency 9 hours after inoculation. To avoid excessive foaming of the culture, an antifoam agent solution is added as needed during the fermentation process. Dissolved oxygen is controlled by a stirred cascade with an oxygen sparge as needed. The culture pH is maintained using acid and base addition. The growth medium is preferably maintained at 36 - 38°C and pH 7.0 - 7.4 by stirring and aeration.

[0082] The nutrient feed consists of yeast extract, Martone B-1, L-cysteine HCl, and glycerol. The feed is initiated when the glucose concentration drops below 10 g / L (12 - 14 hours after inoculation) and continues at a constant rate until production ends. Expression occurs upon addition of IPTG. Induction continues for 18 hours. Following completion of the fermentation process, a cool-down is performed in preparation for the harvest operation. The production fermenter yields a titer of approximately 6 g / L of soluble arginase 1. An overview of the production fermentation is shown in Figure 3.

[0083] Harvest operation The harvest operation captures cells containing soluble arginase 1, disrupts and lyses the cells, and removes the lysate of cell debris using centrifugation and / or filtration. The recovered cell slurry can be frozen or stored at low temperature for long-term storage. The harvest operation can collect cells by centrifugation, pass them through a homogenizer or cell disruption under pressure (French press) twice to lyse, perform a second centrifugation, and filter through a membrane prior to the first chromatography step.

[0084] In a preferred embodiment, all cells are separated from the fermentation broth using a disk stack centrifuge. The resulting cell slurry is resuspended in 25 mM HEPES, pH 7.6, and passed through a homogenizer twice. The pH of 25 mM HEPES can also be used in the range of pH 7.2 - 7.6. The lysed material is clarified using a centrifuge to remove cell debris and then membrane filtered through a 0.2 μm grade filter. In a preferred embodiment, the harvesting step is carried out at a target temperature of ≤15°C.

[0085] In an alternative embodiment, cell disruption is carried out using high pressure. The cell slurry is transferred to a homogenizer at a controlled rate and the homogenized effluent passes through a heat exchanger to reduce the temperature rise seen during pressure homogenization. The cooled cells undergo two passes of homogenization. The lysate pool from the first pass is returned from the collection vessel to the feed vessel. The holding period between passes is minimized to reduce potential microbial growth.

[0086] The material after lysis is clarified by centrifugation to remove cell debris from the soluble components of the lysate. The lysate is transferred to a disk stack, intermittent discharge, centrifuge at a controlled rate. The clarified lysate is collected for further processing.

[0087] Filter the clarified lysate with a ~0.2 μm filter or the like. A process transition filter can also be used for microbial control during process operation. For this purpose, the filter can be a 0.5 μm or 0.2 μm filter. In this step, small particles that may not have been separated during the clarification operation are also removed from the clarified material. Before use, the filter is thoroughly rinsed with purified water and equilibrated with 25 mM HEPES, pH 7.6 buffer. A prefilter can be attached before each downstream process step to reduce the possibility of bioburden carryover.

[0088] Purification of rhARG1, Co-rhARG1, and Co-rhARG1-PEG Regardless of the method used to culture cells expressing rhARG1 (e.g., the fermentation process described above), rhARG1 can be captured and the enzyme further purified using the purification methods described herein. The purification method can include optional steps such as loading cobalt to produce Co-rhARG1 and / or reacting with a PEGylated reactant to produce Co-rhARG1-PEG.

[0089] Various embodiments of the purification process relate to 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") of a system comprising a plurality of chromatography columns. The protein product eluted from this Column 1 is the "first protein product".

[0090] In one or more embodiments, Column 1 uses cation exchange chromatography to bind rhARG1 at a pH in the range of about 7 to about 8, such as a pH of about 7.6. In one or more embodiments, rhARG1 binds in the absence or at low salt concentration. In one or more embodiments, rhARG1 is eluted with a buffer at a high salt concentration (such as NaCl) of 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.

[0091] In various embodiments, a salt gradient is used to separate different charge variants of rhARG1. Exemplary salt gradients are about 0 to about 0.5 M NaCl, about 0 to about 0.4 M NaCl, about 0 to about 0.3 M NaCl, about 0 to about 0.2 M NaCl, or about 0 to about 0.1 M NaCl.

[0092] In one or more embodiments, the method further comprises loading a 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 (“the second protein product”). In another embodiment, the method further comprises loading the second protein product onto a third column that captures and then elutes arginase 1 to provide a third protein product (“the third protein product”). In some embodiments, this third chromatography column (“Column 3”) can be a size exclusion chromatography (SEC) column or a multimodal chromatography (MMC) column.

[0093] Various embodiments provide that for rhARG1, Co is carried to replace the Mn cofactor. In one or more embodiments, the Co loading is carried out using a Co2+ salt, such as CoCl2. Since the incubation time depends on the temperature, the lower the cobalt substitution temperature, the longer the incubation time, and the higher the cobalt substitution temperature, the less long the incubation time is required. The cobalt loading temperature can be as low as about 1 °C or exceed 50 °C, and the corresponding incubation time can be 8 hours or more or less than 10 minutes.

[0094] Various embodiments provide that rhARG1 or Co-rhARG1 reacts with a PEGylation reactant such as methoxy PEG succinimidyl carboxymethyl ester (MW 5000). The PEGylation reactant is typically provided in a 10 - 40 molar excess compared to the enzyme. The incubation time can range from 0.5 to 4 hours. The pH during PEGylation can be from about 8 to about 9, such as a pH of about 8.4.

[0095] In one or more embodiments, the purified PEGylated protein, rhARG1 or Co-rhARG1, comprises arginase I monomer, glucosylenated arginase I, phosphoglucosylenated arginase I, 2× glucosylenated arginase I, glucosylenated + phosphoglucosylenated arginase I, and 2× phosphoglucosylenated arginase I.

[0096] In some embodiments, rhARG1 or Co-rhARG1, which is a purified PEGylated protein, comprises at least 70% arginase I monomer. Exemplary amounts include at least 70%, at least 75%, at least 80%, at least 85%, or at least 90% arginase I monomer. In some embodiments, rhARG1 or Co-rhARG1, which is a purified PEGylated protein, comprises less than 10% glucosylenated arginase I. Exemplary amounts include about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, or about 9% glucosylenated arginase I, or any range between these values. In some embodiments, rhARG1 or Co-rhARG1, which is a purified PEGylated protein, comprises less than 10% phosphoglucosylenated arginase I. Exemplary amounts include about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, or about 9% phosphoglucosylenated arginase I. In some embodiments, rhARG1 or Co-rhARG1, which is a purified PEGylated protein, comprises at least 70% arginase I monomer, less than 10% glucosylenated arginase I, and less than 10% phosphoglucosylenated arginase I.

[0097] Imaged capillary isoelectric focusing (iCIEF) provides a measure of the consistency of a pegylated protein based on the level of heterogeneity of the PEGylated trimer. In one or more embodiments, the icIEF analysis of purified PEGylated proteins, rhARG1 or Co-rhARG1, are nine distinct peaks corresponding to nine distinct charged species, species-1, species-2, species-3, species-4, species-5, species-6, species-7, species-8, species-9, respectively: peak 1, peak 2, peak 3, peak 4, peak 5, peak 6, peak -7, peak -8, and peak -9. The area under the curve of each peak corresponds to the percentage of that particular species. In one or more embodiments, specific peaks can be combined together for related species such as peak 1+2 or peak 3+4.

[0098] In some embodiments, the purified PEGylated proteins, rhARG1 or Co-rhARG1, include the area under the curve of peak 1 at a percentage of less than about 30%, less than about 25%, less than about 20%, less than about 15%, and less than about 10%.

[0099] In some embodiments, the purified PEGylated proteins, rhARG1 or Co-rhARG1, include the area under the curve of peak 2 at a percentage 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%.

[0100] In some embodiments, the purified PEGylated proteins, rhARG1 or Co-rhARG1, include the area under the curve of the combined peak 1+2 at a percentage 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%.

[0101] In some embodiments, the purified PEGylated protein, rhARG1 or Co-rhARG1, comprises the area under the curve of peaks 3 + 4 in a ratio range of about 2% to about 40%, about 2% to about 35%, about 2% to about 30%, about 2% to about 25%, about 4% to about 40%, about 4% to about 35%, about 4% to about 30%, about 4% to about 25%, about 6% to about 40%, about 6% to about 35%, about 6% to about 30%, about 6% to about 25%, about 8% to about 40%, about 8% to about 35%, about 8% to about 30%, about 8% to about 25%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, and about 10% to about 25%.

[0102] In some embodiments, the purified PEGylated protein, rhARG1 or Co-rhARG1, comprises the area under the curve of peak 5 in a ratio range of about 5% to about 40%, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 10% to about 40%, about 10% to about 35%, 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%.

[0103] In some embodiments, the purified PEGylated protein, rhARG1 or Co-rhARG1, comprises the area under the curve of peak 6 in a ratio range of about 2% to about 35%, about 2% to about 30%, about 2% to about 25%, about 2% to about 20%, about 4% to about 35%, about 4% to about 30%, about 4% to about 25%, about 4% to about 20%, about 6% to about 35%, about 6% to about 30%, about 6% to about 25%, about 6% to about 20%, about 8% to about 35%, about 8% to about 30%, about 8% to about 25%, about 8% to about 20%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, and about 10% to about 20%.

[0104] In some embodiments, the purified PEGylated protein, rhARG1 or Co-rhARG1, comprises the area under the curve of peak 7 in a ratio 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%.

[0105] In some embodiments, the purified PEGylated protein, rhARG1 or Co-rhARG1, comprises the area under the curve of peak 8 at a ratio of less than about 25%, less than about 20%, less than about 15%, less than about 10%, and less than about 5%.

[0106] In some embodiments, the purified PEGylated protein, rhARG1 or Co-rhARG1, comprises the area under the curve of peak 9 at a ratio 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%.

[0107] Administration of rhARG1, Co-rhARG1, and Co-rhARG1-PEG The rhARG1, Co-rhARG1, and Co-rhARG1-PEG (and compositions containing them) described herein 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 (or compositions containing them) are administered intravenously (IV) or subcutaneously (SC). Compositions containing rhARG1, Co-rhARG1, and Co-rhARG1-PEG can be provided in formulations with physiologically acceptable liquid, gel, or solid carriers, diluents, and excipients. Such compositions are typically prepared as liquid solutions or suspensions for injection. Suitable diluents and excipients include, for example, water, saline, dextrose, or glycerol, and combinations thereof. Additionally, if desired, the composition may contain small amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH buffering agents.

[0108] Exemplary methods and explanations regarding the administration of rhARG1, Co-rhARG1, and Co-rhARG1-PEG (e.g., pegloticase) are provided below. The following description is specific to pegloticase, but the methods and explanations are applicable to other recombinant arginase 1 and 2 enzymes as well.

[0109] Recommended intravenous dosing regimen: Before starting treatment, obtain the baseline plasma arginine concentration. The initial recommended dose of pegzilarginase for ARG1-D patients administered once weekly as a single intravenous injection is 0.10 mg / kg. If plasma arginine does not decrease to a level of ≤150 micromol / L with an initial dose of 0.10 mg / kg, the dose can be adjusted up to a maximum of 0.20 mg / kg once weekly. If the plasma arginine level falls below 50 micromol / L during treatment, consider reducing the dose. If pegzilarginase has been administered intravenously more than 5 times, consider using subcutaneous administration of pegzilarginase in ARG1-D patients and continue to monitor plasma arginine levels regularly.

[0110] Recommended subcutaneous dosing regimen: When transitioning from intravenous to subcutaneous administration of pegzilarginase, perform the first subcutaneous administration in place of the next scheduled intravenous administration. The initial subcutaneous dose should be the same mg / kg dose as the last IV dose administered. The subcutaneous dose can be adjusted clinically as indicated to keep the plasma arginine level within the range of 50 - 150 micromol / L.

[0111] Arginine blood monitoring: After starting treatment with pegzilarginase, plasma arginine monitoring is required until the patient's plasma arginine level is within the target range of 50 - 150 μmol / L. Thereafter, regular plasma arginine monitoring is recommended to evaluate arginine blood control. Additional plasma arginine monitoring may be required when changing to subcutaneous administration or when changing the diet.

[0112] Instructions for preparation and administration Pegzilarginase is supplied as a frozen liquid formulation in a 10 mL disposable glass vial containing 5 mL of pegzilarginase at a concentration of 1 mg / mL or 5 mg / mL. Each disposable glass vial of pegzilarginase is intended for use as a single intravenous or subcutaneous injection. Before administration, visually inspect pegzilarginase for the absence of particulate matter and discoloration. Pegzilarginase is a colorless to slightly yellow or slightly pink solution. Discard if discoloration, cloudiness, or particulate matter is present in the vial. Remove the flip-top from the vial. Disinfect the rubber stopper of the vial by wiping it with an alcohol swab. Using a sterile syringe with an 18G needle, withdraw the appropriate amount of drug from the vial. If multiple vials are required, a separate needle should be used to withdraw solution from each vial. Calculate the volume of solution to withdraw from the vial for use with a syringe pump. Once the appropriate amount of drug has been drawn into the syringe, use a different needle to draw in normal saline so that the total volume is 40 mL. Calculate the required amount of drug to use as follows: Amount of 1.0 mg / mL pegzilarginase = Patient's weight (kg) × Dose level (mg / kg) Amount of 5.0 mg / mL pegzilarginase = Patient's weight (kg) × Dose level (mg / kg)

[0113] Administer pegzilarginase by intravenous infusion over 30 minutes or more using a syringe pump.

Table 3

[0114] 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 volume of 1 mL / injection for pediatric patients. If the calculated volume for subcutaneous administration is greater than the maximum volume, a higher vial concentration can be used (e.g., 5 mg / mL instead of 1 mg / mL) or the volume can be divided into multiple smaller injections (e.g., a 4 mL injection is divided into two 2 mL injections).

[0115] Dosage Form and Strength Pegzilarginase injection is available in 10 mL vials as a colorless to slightly yellow or slightly pink solution and is available as follows: Available in 10 mL vials as follows: a. Solution for injection: 5 mL of 1.0 mg / mL b. Solution for injection: 5 mL of 5.0 mg / mL

[0116] Warnings and Precautions An allergic reaction may occur upon administration of pegzilarginase. Monitor all patients for signs and symptoms of acute allergic reactions (urticaria, pruritus, erythema, hypotension, tachycardia, etc.) during and after pegzilarginase infusion. In case of severe allergic reaction, immediately delay or discontinue the administration of pegzilarginase and provide appropriate medical treatment. Consider premedication of patients with a sedation-free antihistamine prior to dosing. If corticosteroids are required, they should be used with caution as they may cause hyperammonemia.

[0117] Pregnancy: Pregnancy Category B Reproductive studies have been conducted in mice and rats at doses up to 100 mg / kg. There was no evidence of harm to the fetus due to pegzilarginase. However, there are no adequate and well-controlled studies in pregnant women. Since animal reproductive studies are not always predictive of human response, pegzilarginase should be used during pregnancy only if clearly needed.

[0118] Nursing Mothers It is not known whether pegzilarginase is present in human milk. The developmental and health benefits of breastfeeding should be considered along with the mother's clinical need for pegzilarginase and any potential adverse effects of the drug on the breastfed infant.

[0119] Description Pegargi-nase is a cobalt-substituted recombinant human arginase I enzyme that is covalently conjugated to monomethoxypolyethylene glycol (mPEG) and functions to catalyze the same reaction as arginase 1, converting arginine to ornithine and urea. Human arginase 1 is a binuclear manganese metalloenzyme. To produce pegargi-nase, the manganese cofactor is replaced with cobalt to generate Co-arginase I. Substituting the native manganese (Mn+2) with cobalt (Co+2) at the active site of arginase I improves stability and catalytic activity at physiological pH. Pegylation extends the circulation half-life. The average molecular weight of pegargi-nase is approximately 284 kDa. Pegargi-nase has a specific activity in the range of approximately 320 - 600 units per mg of protein content. One activity unit is defined as the amount of enzyme required to convert 1 micromole of arginine to ornithine per minute at 37°C.

[0120] Pegargi-nase is intended for intravenous or subcutaneous injection and is supplied as a sterile, clear, colorless to slightly yellow or slightly pink solution formulated in a buffer at concentrations of 1 mg / mL and 5 mg / mL, containing 50 mM sodium chloride, 5 mM potassium phosphate, and 1.5% w / v glycerol, at pH 7.4. It is provided as a preservative-free sterile solution in a clear disposable glass vial. Each vial of the 1 mg / mL pegargi-nase formulation contains 5 mL of the formulation (5 mg of pegargi-nase per vial). Each vial of the 5 mg / mL pegargi-nase formulation contains 5 mL of the formulation (25 mg of pegargi-nase per vial). The vials are stoppered with a coated rubber stopper, sealed with an aluminum flip-off seal, stored frozen at ≤ -60°C, and thawed before use.

[0121] Pharmacodynamics In adult and pediatric patients with arginase 1 deficiency, pegargnase treatment reduced blood arginine concentration from pre-treatment baseline values to the normal blood arginine range of 40 - 115 μmol / L. The maximum suppression of L-arginine was observed approximately 8 hours after dosing, decreased in a dose-dependent manner, and recovered to pre-dose levels by 168 hours after dosing. A strong correlation was observed between pegargnase and arginine, with an immediate inhibitory effect on arginine after IV administration and reaching the maximum decrease in arginine concentration within 24 hours after dosing.

[0122] Pharmacokinetics: After IV administration to 14 subjects, pharmacokinetic samples were collected throughout the 0 - 168 hour dosing interval to characterize the relationship between the pharmacokinetics of pegargnase and arginine. Across the dose range (0.015 mg / kg - 0.2 mg / kg), pegargnase exposure measured by Cmax and AUC0 - 168 increased approximately proportionally to the dose, with a 13-fold increase in dose resulting in a 14-fold increase in Cmax and AUC0 - 168. Accumulation of pegargnase was not observed after a once-weekly IV dosing regimen, with a T1 / 2 of approximately 30 hours across the dose range and low to moderate inter-subject variability (13 - 46% CV) in exposure measurements.

[0123] Animal Toxicology and / or Pharmacology The pharmacological effect of pegargnase on arginine levels was evaluated in a neonatal transgenic mouse model of arginase I and a tamoxifen-induced arginase deficiency model in adult mice. These models mimic human disease in that circulating arginine and arginine catabolites are present in substantial excess, although unlike humans with arginase I deficiency, these animals develop severe and generally fatal hyperammonemia. The pharmacological effect was also evaluated in a rat arginine-induced model of hyperargininemia. Pegargnase decreased plasma arginine levels in a dose-dependent manner.

[0124] The potential toxicity of pegylated arginase and TK was evaluated in juvenile rats (equivalent to 2-year-old humans) on postnatal day (PND) 21 by administering 0.1, 0.3, and 1.0 mg / kg of an IV bolus injection once a week for 6 months, followed by a 6-week recovery period. Pegylated arginase was well tolerated, with no mortality related to the test substance, and no significant test substance effects were observed on food intake, coagulation, urine tests, ophthalmoscopic examinations, sexual maturation, growth hormone analysis, bone marrow analysis, functional observation battery evaluation (FOB), or neurobehavioral tests (auditory startle habituation, locomotor activity, or Morris water maze). No pegylated arginase-related macroscopic findings were present at the end of 6 months and at the end of the 6-week recovery interval. Adverse microscopic changes were limited to the testes and epididymides and were correlated with a decrease in male genital weight and adverse sperm analysis findings at 0.3 and 1.0 mg / kg. At 1.0 mg / kg, adverse effects on sperm analysis were seen, such as a decrease in sperm motility, a decrease in the number of sperm in the caudal epididymides, a decrease in sperm concentration, and an increase in the proportion of abnormal sperm. These observations were considered direct treatment-related effects and were correlated with microscopic changes of subtle tubule degeneration in the testes at 0.3 and 1.0 mg / kg. After the 6-week recovery period in the control and 1.0 mg / kg groups, these changes were generally reversible, except for an increase in the proportion of abnormal sperm and sperm number. Since the normal sperm development cycle is about 9 weeks or longer than the 6-week recovery period, the partial reversibility after 6 weeks was not unexpected.

[0125] Importantly, no obvious PEGylation effects were observed by histopathology. Toxicokinetic data indicated that pegylated arginase exposure was maintained throughout the study. As a conclusion, the NOAEL for females was 1.0 mg / kg. In males, based on microscopic changes in the testes at 0.3 and 1.0 mg / kg, the NOAEL was 0.1 mg / kg.

[0126] The potential toxicity of pegzilarginase and TK was evaluated in cynomolgus monkeys by intravenous bolus injection once a week for 13 weeks at doses of 0.1, 0.3, and 1.0 mg / kg, followed by a 4-week recovery period. Clinical symptoms observed at 1.0 mg / kg included weight loss, increased incidence of sparse hair (general), dryness / discoloration of the skin (general), tremors, anorexia, watery stools, decreased activity, ataxia, muscle wasting, and / or a scruffy / round-backed appearance. No treatment-related effects were observed in clinical pathology parameters (coagulation, growth hormone, and urine tests), ECG and ophthalmic examinations, respiratory rate, and blood pressure evaluations.

[0127] Supply method / Storage and handling Pegzilarginase is provided as an injectable solution.

[0128] Pegzilarginase is supplied in a frozen state (≤ -60°C). Diluted pegzilarginase should be used immediately. If it cannot be used immediately, diluted pegzilarginase can be stored at 2°C to 8°C (36°F to 46°F) for a maximum of 8 hours during administration.

Example

[0129] Before describing some exemplary embodiments of the present disclosure, it should be understood that the present disclosure is not limited to the details of the construction or process steps described hereinafter in the following description. The present disclosure is capable of other embodiments and can be practiced or implemented in various ways.

[0130] In the following experimental disclosure, the following abbreviations apply: eq (equivalent), M (mole), μM (micromole), mM (millimole), N (normal), mol (mole), mmol (millimole), μmol (micromole), nmol (nanomole), 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).

[0131] Example 1: Cation Exchange Column Chromatography (Column 1) In a preferred embodiment, arginase 1 is captured on a cation exchange column (CEX) to reduce product - related impurities such as host cell proteins (HCP), DNA, and endotoxin, and process - related impurities (see Figure 3 for an overview of the purification process). In a particular embodiment, the first column (Column 1) chromatography step of the arginase 1 purification process uses an SP Sepharose FF resin and an inlet heat exchanger. Column 1 uses cation exchange chromatography to bind arginase 1 in the absence of salt at pH 7.6 and elutes with a buffer with increased salt concentration (Figure 4(a)). In one embodiment, the salt is NaCl and the elution from Column 1 is performed at room temperature using 25 mM HEPES, 0.1 M NaCl, pH 7.2 - 7.6. However, alternative embodiments such as the application of an NaCl gradient to Column 1 are possible.

[0132] Figure 4(a) shows a representative purification of arginase 1 on Column 1. Approximately 3 liters of clarified E. Coli lysate was loaded onto a cation exchange column. As can be seen from the high level of absorbance at 280 nm, a large amount of protein does not bind to the column and is detected in the flow - through. Next, the column was washed with approximately 2 liters of column wash buffer. The fraction in which arginase 1 was concentrated was eluted with 0.1 M NaCl (final peak), as detected by absorbance at 280 nm.

[0133] Example 2: Cobalt Substitution In a preferred embodiment, the arginase 1 native manganese coenzyme is replaced by cobalt. During cobalt substitution (also called cobalt loading), one or both of the two manganese ions normally present in arginase 1 are replaced by cobalt ions. Various temperatures can be used for the cobalt substitution step, and a wide range of cobalt concentrations can also be used (see Table 2). The incubation time for cobalt substitution is as short as 10 minutes and can be carried out at 50 °C or higher. Conversely, the cobalt loading temperature is as low as 1 °C or 5 °C and can be carried out for more than 8 hours. Also, the higher the proportion of cobalt loaded on arginase 1, the higher the specific activity.

[0134] The arginase 1 eluted from column 1 (also called column 1 pool) can be held at room temperature for the cobalt substitution step. In one embodiment, the stock cobalt chloride solution (0.5 M CoCl2) is diluted 50-fold by adding it to the column 1 pool at a defined rate, and the final cobalt chloride concentration is 10 mM. Next, the cobalt substitution is mixed at 20 °C for 2 hours. In another embodiment, the arginase 1 cobalt loading is carried out in a 10 mM CoCl2 solution at room temperature for about 2 hours to about 8 hours.

[0135] An overview of the cobalt loading step is shown in Table 3.

Table 4

[0136] Example 3: Ultrafiltration / Diafiltration 1 (UF / DF1) UF / DF1 removes free cobalt ions and exchanges Co - arginase 1 into the solution in preparation for anion - exchange chromatography. In the UF / DF1 step, a membrane with a molecular weight cut - off of 30 kDa is used. One of the important functions of this step is to lower the level of free cobalt and perform buffer exchange of the Co - arginase 1 pool prior to anion - exchange chromatography. The membrane is disinfected with a wash solution (0.5 N NaOH) and rinsed with water. A normalized water permeability test (NWP) is performed and then the membrane is equilibrated before use in production. Once the UF / DF system is equilibrated, the Co - arginase 1 pool is diafiltered against 3 diavolumes of 25 mM HEPES, 0.1 M NaCl, pH 7.6, followed by 4 diavolumes of 50 mM Tris, pH 8.4. After diafiltration, the pool is recycled and recovered from the system using 2 times the system hold - up volume of 50 mM Tris, pH 8.4.

[0137] The UF / DF1 membrane is washed by performing a denaturing wash step where it is recirculated with 0.5 N NaOH for 30 minutes after a 2 M NaCl flush. The system is rinsed with purified water and the NWP is tested to evaluate the effectiveness of the wash procedure. The membrane can be stored in 0.1 N NaOH.

[0138] In an alternative embodiment, the first buffer exchange is to 25 mM HEPES, 0.1 M NaCl, pH 7.2 - 7.6 and the second buffer exchange is to 50 mM Tris, pH 8.1 - 8.5.

[0139] Example 4: Anion - column chromatography (Column 2) A preferred embodiment of arginase 1 purification uses another column (“Column 2”) which is anion exchange column chromatography. One embodiment of Column 2 is Q Sepharose FF resin. One function of this Column 2 step is to reduce process-related impurities such as host cell DNA and endotoxin from the UF / DF1 pool. Column 2 binds these impurities while Co-arginase 1 flows through and is collected in the column effluent during the loading and washing 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 resin is loaded onto the column having buffer 50 mM Tris, pH 8.1 - 8.5.

[0140] In another embodiment of the method, while arginase 1 is being recovered in the flow-through, the first protein product is loaded onto an anion exchange column to capture impurities. Figure 4(b) is a representative chromatogram of arginase 1 purification on an anion exchange column (Column 2). As can be seen from the absorbance at 280 nm, a large amount of protein is detected in the flow-through. The impurities are captured by Column 2 and not eluted into the Column 2 pool (also called the second protein product), and arginase 1 is further concentrated.

[0141] Example 5: Capto multimodal column chromatography (Column 3) In a preferred embodiment, the arginase purification process uses a third column chromatography column (Column 3). In one embodiment, Column 3 is a Capto multimodal chromatography (MMC) column or a size exclusion column. Embodiments using MMC capture arginase 1 on the column while process-related impurities such as host cell proteins (HCPs), DNA, and endotoxins are washed away in the flow-through. In this embodiment, Co-arginase 1 can be captured by the column in the absence of salt at pH 8.4, and then Co-arginase 1 can be eluted with a buffer of increased salt concentration. A representative example of a Capto Multimodal Cation Exchange chromatography column is shown in Figure 4(c).

[0142] In one embodiment, the MMC chromatography (Column 3) uses approximately 15 column volumes, loaded with up to 30 g of protein / L resin, and the high-salt concentration step elution is performed at 50 mM Tris, 250 mM NaCl, pH 8.1 - 8.5. In some 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 the bound Co-arginase 1 is eluted using 50 mM tromethamine and 250 mM sodium chloride.

[0143] Example 6: Ultrafiltration / Diafiltration 2 (UF / DF2) UF / DF2 concentrates arginase 1 and exchanges the protein into the pre-PEGylated intermediate. In the UF / DF2 step, a membrane with a molecular weight cut-off of 30 kDa is used. An important function of this step is to perform a buffer exchange on the column 3 pool of the non-PEGylated Co-arginase 1 intermediate prior to PEGylation (or prior to additional filtration and storage). The membrane is disinfected with a cleaning solution (0.5 N NaOH) and rinsed with water. Once the UF / DF system is equilibrated, the column 3 pool (also called the third protein product) is diafiltered against 20 mM sodium phosphate, 50 mM sodium chloride, 1.5% (w / v) glycerol, pH 7.4 with a five-fold diavolumes. If the pool concentration of column 3 is <8 g / l, the pool is further concentrated to 8 g / L. After diafiltration (and concentration if necessary), the pool is recycled and recovered from the system using twice the hold-up volume of the system with 20 mM sodium phosphate, 50 mM sodium chloride, 1.5% (w / v) glycerol, pH 7.4. After recovery, a two-step dilution with the diafiltration solution can be used. The concentration target for the first dilution is 6 g / L and the concentration target for the second step is 5 g / L. Two steps can be used to reach the target. If the concentration after the first dilution is within the target range, the second step may not be necessary.

[0144] Example 7: Intermediate Filtration and UF / DF3 Prior to the PEGylation reaction, cobalt-containing arginase 1 can be stored long-term, including long-term freezing. The intermediate Co-arginase can be filtered through a 0.2 μm filter and frozen for long-term storage.

[0145] In the UF / DF3 step, a membrane with a molecular weight cut-off of 30 kDa is used. One function of this step is to perform buffer exchange and concentrate the filtered UF / DF2 pool (fresh or thawed) to provide optimal conditions for PEGylation. When using the frozen Co-arginase 1 intermediate as the starting material, thawing is carried out at room temperature for a maximum of 36 hours. The membrane is disinfected with a cleaning solution (0.5 N NaOH) and rinsed with water. A standardized water permeability test (NWP) is performed and then equilibrated before use in production. Once the UF / DF system is equilibrated, the Co-arginase 1 intermediate is diafiltered with five diavolumes against 0.1 M sodium phosphate (pH 8.4). After diafiltration, the pool is concentrated, recycled, and recovered from the system using twice the system hold-up volume of 0.1 M sodium phosphate, pH 8.4. After recovery, it is diluted in two steps with the diafiltration solution. The concentration target for the first dilution is 11 g / L, and the concentration target for the second step is 10 g / L. Two steps are utilized to facilitate the target being at the level. If the concentration after the first dilution is within the target range, the second step may not be necessary.

[0146] Regarding the steps of UF / DF2 and UF / DF3, the first buffer exchange can be 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 is concentrated to approximately 10.0 mg / mL (in the preparation for PEGylation of the active pharmaceutical ingredient).

[0147] Example 8: PEGylation of Arginase 1 PEGylation involves covalently attaching PEG (polyethylene glycol) to the Co-arginase 1 (drug substance) molecule (see Table 4 for representative embodiments of the PEGylation step). In one embodiment, the PEGylation reaction covalently attaches a 5000 Da PEG molecule to Co-arginase 1. In an alternative embodiment, PEGylation can be performed before the cobalt substitution of arginase 1 or at other points in the production process. In one embodiment, for the PEG conjugation reaction, a solid or liquid methoxy PEG succinimidyl carboxymethyl ester that reacts with the sterically available lysines on Co-arginase 1 can be used. The molecular weight of the resulting PEGylated protein (Co-rhARG1-PEG) is approximately 280 kDa. The PEGylated pool is filtered and can be stored at 2 - 8 °C up to the UF / DF4 operation.

Table 5

[0148] In one embodiment, solid methoxy PEG succinimidyl carboxymethyl ester (MW 5000) can be added to the arginase 1-containing solution in a 19.3-fold molar excess and incubated at pH 8.4 for 0.5 - 4.0 hours.

[0149] Following PEGylation, ultrafiltration / diafiltration is used to remove unbound PEG, exchange arginase 1 into the formulation buffer, and concentrate arginase 1 for the formulation step. In this UF / DF4 step, a membrane with a molecular weight cut-off of 100 kDa is used. One function of this step is to buffer-exchange the PEG pool into the final formulation while removing free PEG. The membrane used for this purpose is sterilized with a wash solution (0.5 N NaOH) and rinsed with water. Once the UF / DF system is equilibrated, the PEG pool is diafiltered against 5 mM potassium phosphate, 50 mM sodium chloride, 1.5% (w / v) glycerol, pH 7.4 at 10 diavolumes. After diafiltration, the pool is recovered from the system by applying pressure. The recovered UF / DF4 pool is diluted to 5 g / L with 5 mM potassium phosphate, 50 mM sodium chloride, 1.5% (w / v) glycerol, pH 7.4 prior to the final filtration and filling steps. In an alternative embodiment, arginase 1 is 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.

[0150] In some embodiments, the formulation buffer, 5 mM potassium phosphate, 50 mM sodium chloride, 1.5% glycerol, pH 7.4 has been found to enhance the stability of arginase 1 upon storage as compared to other buffers such as sodium phosphate buffer. In one or more embodiments, the buffer 5 mM potassium phosphate comprises 1 mM K2HPO4 and 4 mM KH2PO4.

[0151] The original drug (Co-rhARG1-PEG) is a PEGylated cobalt-substituted human arginase 1 created by conjugating activated PEG molecules to the ε-amino group of lysine and the amine group of the N-terminal amino acid. An orthophthalaldehyde-based fluorescent assay is used to determine the molar ratio of PEG molecules per protein using orthophthalaldehyde. Orthophthalaldehyde reacts with primary amines, especially in the presence of thiols, to form fluorescent derivatives. Measurement of the fluorescent signal enables 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 the fluorescent assay of the PEGylated original drug from the theoretical number of free amines present in non-conjugated Co-arginase 1. The theoretical number of free amines from lysine residues and the N-terminal amino acid is 25. Free non-conjugated PEG in the original drug is measured by SEC-HPLC with refractive index detection. Results can be expressed as μg / mL of free PEG (see Table 5).

Table 6

[0152] Example 9: CIEX-HPLC Characterization of Drug Intermediates During the fermentation of E. coli, various arginase 1 charge variants can be produced. Charge variants can be analyzed by cation exchange HPLC (CIEX-HPLC) using a TSK gel cation exchange column. In this type of analysis, a mobile phase (A) of 20 mM MES, pH 6.0 and a mobile phase (B) of 20 mM MES, 500 mM NaCl pH 6.0, a flow rate of 1.0 mL / min, a run time of 40.0 minutes, a column temperature of 22 °C, and a mobile phase gradient according to Table 6 are used.

Table 7

[0153] Before analysis, the sample is diluted with formulation buffer. The results are described as percent charge variant distribution. A representative chromatogram is shown in Figure 5(a), and usually six major peaks are observed in the Co arginase 1 intermediate.

[0154] Example 10: Evaluation of the iCIEF Characteristics of the API The drug intermediate is PEGylated to form the API. PEGylation of the drug intermediate makes the use of the drug intermediate CIEX-HPLC method less suitable than other embodiments developed as part of the present invention. Anionic IEX-HPLC was evaluated, but sufficient separation was not obtained. Instead, an imaging capillary isoelectric focusing (iCIEF) method was developed to analyze the charge variants of the API.

[0155] The analyte for imaging capillary isoelectric focusing (iCIEF) moves within the capillary by the counter-movement of hydronium ions (anolyte) and hydroxyl ions (catholyte) in the presence of an electric field. The sample is diluted in a matrix containing carrier ampholytes and pI markers. Protein separation is performed in two focusing steps. The first prefocusing step establishes a pH gradient. Charge variants are more sharply focused and separated during the second, higher voltage focusing step. Images of the UV light absorption across the capillary are digitally captured every 30 seconds and after completion of the focusing step.

[0156] The results can be expressed as percent charge variant distribution. A representative electrophoresis diagram is shown in Figure 5(b), and nine major peaks are observed for the API. Peaks 3 and 4 are integrated together because the resolution between these peaks has been shown to be variable. The relative areas of these peaks are shown in Table 7.

Table 8

[0157] Example 11: Enzymatic Activity of Co-Arginase 1 Intermediate and API The enzymatic assay used to measure the activity and establish the identity of the Co-arginase 1 intermediate and Co-rhARG1-PEG API monitors the conversion of arginine to ornithine. The reaction mixture has one enzyme concentration tested at seven different arginine substrate concentrations in the range of 0 - 2 mM. The reaction is carried out at 37 °C for a certain period of time. The reaction time is set so that the consumption of the substrate is less than 10% at any substrate concentration. The reaction is quenched, the product ornithine is derivatized, and quantified by reverse-phase UPLC.

[0183] Examples of plots of reaction rate vs. substrate concentration are shown in Fig. 8(a) (Co-arginase 1 intermediate) and Fig. 8(b) (Co-rhARG1-PEG API) along with representative Kcat, Km, and Kcat / Km values.

[0158] Example 12: Analysis of Cobalt and Manganese Cobalt, residual manganese, and free cobalt were measured using inductively coupled plasma mass spectrometry (ICP-MS). Samples were decomposed by microwave and all metals were released from the matrix using 1% nitric acid and 6% hydrogen peroxide. The resulting digest was analyzed by ICP-MS. Samples of cobalt and residual manganese were decomposed without sample treatment. To measure cobalt not bound to the enzyme, free cobalt was measured for the filtrate sample that had been ultrafiltered to separate the enzyme from the filtrate. Free cobalt was measured. Table 8 summarizes some of the characteristics of the Co-arginase 1 intermediate.

Table 9

Table 10

[0159] Example 13: Post-Translational Modification of Co-Arginase 1 Intermediate Post-translational modifications of co-arginase 1 were detected using various techniques such as peptide mapping, LC-MS intact mass analysis, and reverse-phase LC / MS. A summary of all the identified modifications is shown in Table 10.

Table 11

[0160] By characterization, it was found that if there are modifications in the co-arginase 1 intermediate the main modification is N-terminal glucosylation (confirmed by peptide mapping). Additional characterization of the arginase 1 modified species was performed by testing samples taken at three time points (fermentation, post-column 1, and drug intermediate from column 3). In the analytical method, it is usually necessary to separate arginase and analyze it as a monomer. The N-terminal glucosylation of arginase 1 (analyzed as a monomer) was usually 10.8 - 13.9%. Other modifications were N-terminal phosphoglucosylation monomer (4.3 - 6.5%) and diglucosylation monomer (0.7 - 1.2%) across the samples at the three time points. In the samples used from the execution of the standard reference generation, the levels of unmodified co-arginase 1 (monomer) and co-arginase 1 intermediate were equivalent at 80.6% - 83.6% respectively. The standard conditions used in the purification process (i.e., no salt gradient applied to column 1) moderately changed the relative levels of the unmodified monomer that persisted up to the co-arginase 1 intermediate (81.1 - 83.6%).

Table 12

[0161] Example 14: Change in the conditions of column 1 In an alternative embodiment, a NaCl gradient can be applied to column 1. Using a NaCl gradient in column 1 allows for the separation of various arginase 1 variants and the selection of preferred embodiments. Figure 7 shows a gradient of 0.0 - 0.2 M NaCl applied to column 1. Individual fractions collected from the eluate of column 1 were analyzed by SE-HPLC, CEX-HPLC, and RP-HPLC.

[0162] An analytical CEX-HPLC method was used to assign peak numbers from 1 to 6 to the charge variants of arginase 1 (see Figure 5(c)). The peak numbers correspond to various glucosynoylation states and unglucosynoylated arginase 1. This analysis showed six peaks of arginase 1 eluted from the NaCl gradient. The arginase 1 variants assigned peak numbers 1, 2, and 3 eluted early in the elution peak of column 1. Peak 4 eluted from the highest concentration portion of the eluted arginase 1 and peak 5 (unmodified arginase 1), and peak 6 eluted in the latter half of the elution peak. Thus, 0.0 - 0.2 M NaCl successfully separated various charge variants of arginase 1.

[0163] Alternative NaCl gradients such as 0 - 0.5 M NaCl can be used for elution of column 1. It was found that using a NaCl gradient allows arginase 1 to be reproducibly separated into six different peaks, enabling the selection of specific arginase 1 variants for further processing in the manufacture of the drug substance or formulation.

[0164] Further analysis of the first protein product (and arginase 1 variants) was also analyzed by LC / MS (see Figure 6). LC / MS analysis identifies specific types of glucosynoylation generated by the production of arginase 1 in E. coli. LC / MS analysis identified unmodified arginase 1, glucosynoylated arginase 1, phosphoglucosynoylated arginase 1, and 2-fold (2X) glucosynoylated arginase 1.

[0165] Table 12 shows that applying a 0 - 0.2 M NaCl gradient (and the corresponding fractionated CEX peaks 1 - 6) generates fractions with different levels of glucosylation. Each of peaks 1 - 6 was analyzed by LC / MS. The data indicate that the dominant peak (peak 5) has a high proportion of non - glucosylated arginase 1 and a high specific activity. Depending on the desired characteristics, various fractions (corresponding to peaks 1 - 6) can be collected and further processed.

Table 13

[0166] In addition to varying the NaCl concentration in column 1, loading different amounts of protein onto column 1 can enhance the purification of non - glucosylated arginase 1.

[0167] Varying the loading factor of column 1 and using an NaCl gradient in column 1 can compensate for unexpected perturbations that occur during E. coli fermentation producing glucosylated arginase 1.

[0168] Example 15: Changes in Fermentation Conditions Experiments were conducted to determine the robustness of the fermentation conditions for producing arginase 1. Table 13 shows that E. coli fermentation at sub - optimal pH 7.6 produces more glucosylation than fermentation at the preferred pH 7.2. Vessels B1, B8, and B12 used the optimal conditions for fermentation: pH 7.2, dissolved oxygen 30%, and medium feed rate 0.06 mL / min. Vessel B3 was used to ferment arginase 1 - expressing E. coli at pH 7.6 (a higher pH than the optimal conditions). The increase in pH led to a higher proportion of phosphoglucosylation adducts (23% vs. 10 - 12% in the control experiment)

Table 14

[0169] Example 16: Variation in the Loading Factor of Column 1 Varying amounts of E. coli cell lysate were applied to Column 1 to examine the effect on the purification of arginase 1 charge variants and to measure the yield and purity. When loading coefficients of 15 - 60 g of protein / L resin were used under various conditions, a shift in the CIEX charge species profile was shown (Table 14). Higher loading coefficients resulted in better separation of the glucosylated variants (although there may be a trade-off in yield depending on the fraction collected). For example, when the loading coefficient was 20 mg of protein / mL resin, peak 5 was 45.8%, but when the loading coefficient was 40 mg / mL, this increased to 50.0%.

Table 15

[0170] Example 17: Phase 1 / 2 Clinical Trial The formulation produced by the method of the present invention was used in a Phase 1 / 2 open-label study to evaluate the administration of Co-rhARG1-PEG in arginase 1 deficiency and hyperargininemia. The primary endpoint of this study was to evaluate the safety and tolerability of intravenous (IV) administration of Co-rhARG1-PEG in subjects with hyperarginase / arginase 1 deficiency. The secondary endpoints were as follows: to determine the effect of the investigational drug administered IV on plasma arginine concentration, to determine the effect of the investigational drug administered IV on plasma guanidino compounds (GC), and to characterize the pharmacokinetic (PK) profile of the investigational drug administered IV. Other endpoints included the evaluation of clinical outcome measures in obtaining the following clinical benefits: 6-minute walk test (6MWT), Gross Motor Function Measure (GMFM) parts D and E, and Adaptive Behavior Assessment System (ABAS).

[0171] Data from the first half of the phase 1 / 2 study showed that Co-rhARG1-PEG was very effective in continuously reducing plasma arginine. Furthermore, control of the patients' plasma arginine levels was associated with clinically meaningful responses in mobility and adaptive behavior. Tolerance was generally good during treatment. Hypersensitivity reactions were rare and manageable with standard means.

[0172] The Co-rhARG1-PEG formulation supplied for the study was in the form of a liquid formulation in a 10 mL disposable glass vial containing 5 mL of formulated preparation at a concentration of 1 mg / mL. This agent was formulated with 50 mM NaCl, 1 mM K2HPO4, 4 mM KH2PO4, and 1.5% w / v glycerol.

[0173] The first half of the phase 1 / 2 study was conducted in two parts: Part 1 (single ascending dose escalation) and Part 2 (repeated dosing). The study designs of this phase 1 / 2 trial 101A and 102A open-label continuous dosing are shown in the figure below:

Chemical formula

[0174] In Part 1, the drug was introduced to the patients and safety was the focus. In Part 2, the patients were stabilized at a certain dose and designed to search for markers of clinical efficacy. Prior to each part, a baseline assessment of arginine levels was performed. All patients who participated in Part 1 could continue to receive arginase 1 in Part 2 if eligible for continuous dosing.

[0175] In this study, each patient received an escalating starting dose in Part 1, with a 2-week washout / observation period provided between each successive dose level. The possible 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 at 2-week intervals as needed to optimize plasma arginine. If new data from a previous dose level met certain criteria, a particular dose could be repeated or the dose could be increased / decreased between the specified dose levels. For example, dose escalation could stop if one or more of the following dose escalation stop criteria were met: the patient's plasma arginine level was <40 μM continuously for at least 40 (±2) hours post-dose for all samples collected during that period, or the patient's plasma arginine level was on average <115 μM continuously for at least 112 (±2) hours post-dose for all samples collected during that period.

[0176] If none of these events occurred, the patient could escalate to the next higher dose level of arginase 1 every 2 weeks until the dose escalation stop criteria were reached or the maximum dose of 0.30 mg / kg was administered in this protocol. Ultimately, for treatment purposes, the dose could also be increased beyond 0.30 mg / kg.

[0177] Part 2 was the repeat dosing period for patients who completed Part 1. In Part 2, the dose and regimen for each patient were determined, with an emphasis on safely optimizing plasma arginine in the range of approximately 40 μM to approximately 115 μM during repeat dosing and maintaining pre-dose levels below 150 - 200 μM. If the data indicated the potential to better investigate the dose-response results during repeat dosing, it would also have been possible to use several dose levels in Part 2. Arginine levels during treatment were also compared to the arginine levels measured before treatment.

[0178] Patients who completed Part 2 of 101A were eligible to participate in a long-term open-label extension (OLE) trial (NCT03378531). There was an option to switch to subcutaneous administration during the remaining 3-year OLE period after 24 once-weekly intravenous administrations.

[0179] Results Increases in mean Cmax and mean AUC0-168 appeared to be dose-proportional in all patients. Mean (±SD) Cmax was 0.428 ± 0.0915, 0.723 ± 0.247, 1.73 ± 0.538, 2.27 ± 0.238, and 6.13 (N = 1) μg / mL at Co-rhARG1-PEG dose levels of 0.015, 0.03, 0.06, 0.1, and 0.2 mg / kg, respectively. There was a slight anti-drug antibody (ADA) effect on mean Cmax in ADA-positive and ADA-negative patients (Figure 9).

[0180] Changes in AUC (AUC0-168, AUC0-∞) appeared to be dose-proportional across the dose range studied, and no significant change was observed between 0.06 and 0.1 mg / kg (using available data). Estimates of mean clearance (CL) were in the range of 0.789 - 1.57 mL / hour / kg in all patients and 0.776 - 1.33 mL / hour / kg in ADA-negative patients. Estimates of mean volume of distribution (Vss) were in the range of 35.3 - 52.1 mL / kg in all patients and 32.8 - 52.1 mL / kg in ADA-negative patients.

[0181] The first part of the study served to select the optimal (individual) starting dose for each patient in Part 2 using the observed PD (arginine) response. In the first week of Part 2, there was a tendency for the mean circulating drug concentration to increase in all patients in whom the dose of Co-rhARG1-PEG was escalated across the evaluated dose range. After the first administration of Co-rhARG1-PEG in Part 2, the increase in mean Cmax appeared to be proportional to the dose in all patients. The mean (±SD) Cmax was 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 when the dose levels of Co-rhARG1-PEG were 0.015, 0.03, 0.04, 0.06, 0.09, 0.1, and 0.12 mg / kg, respectively.

[0182] In Part 2, at week 8, as the dose of Co-rhARG1-PEG was increased, the mean circulating drug concentration generally increased in all patients. There was no significant ADA effect on the PK concentrations available at week 8. As a result of the data, attainment of steady state was assumed at the current time in most (13 / 14) patients. After the 8th QW administration of Co-rhARG1-PEG, the increases in mean Cmax and AUC0-168 appeared to be proportional to the dose in all patients.

[0183] In addition to the pharmacokinetic data, pharmacodynamic (arginine) data were collected (Figure 10). In patients with arginase 1 deficiency, the QW IV dose of Co-rhARG1-PEG was administered (weekly) in Part 2, and the starting dose was selected based on the PD (arginine) response observed in Part 1. After the first QW IV administration of Co-rhARG1-PEG, a significant decrease in circulating arginine levels was seen, particularly with Co-rhARG1-PEG doses of 0.04 mg / kg or higher. In some cases, individual arginine concentrations fell below 40 μM. Furthermore, at doses of ≥0.04 mg / kg, the recovery to starting arginine levels was incomplete in most patients immediately prior to the second QW (weekly) Co-rhARG1-PEG dose administration.

[0184] Overall, exposure to Co-rhARG1-PEG generally increased, and arginine suppression increased with increasing dose. Individual dose optimization was performed in Part 1, and in weeks 1 and 8 of Part 2, a range of doses with different numbers of patients per dose level were present.

[0185] Example 18: Subcutaneous Administration After completion of Part 2 of the Phase 1 / 2 trial of Example 17, some patients were switched from intravenous administration of Co-rhARG1-PEG to subcutaneous administration. Surprisingly, subcutaneous administration of Co-rhARG1-PEG gave a pharmacodynamic profile that appeared to be superior to IV administration. Also unexpectedly, the same formulation as for intravenous administration was successfully used for subcutaneous administration of Co-rhARG1-PEG.

[0186] Subcutaneous administration of Co-rhARG1-PEG maintained the patients' arginine levels within the preferred (healthy) target range of plasma arginine concentration longer than IV administration (Figure 11). The preferred optimized plasma arginine concentration in patients is in the range of about 40 μM to about 115 μM (during repeated dosing), with emphasis on maintaining levels below 150 - 200 μM prior to dosing. As seen in Figure 11, subcutaneous administration of Co-rhARG1-PEG results in arginine concentrations above the lower limit level of 40 μM and below the upper limit level of 115 μM. Surprisingly, subcutaneous administration gave arginine concentrations that were entirely within the preferred range. This means that patients remained within the appropriate plasma range of arginine concentration until another weekly dose of Co-rhARG1-PEG was administered.

[0187] Example 19: Pharmacodynamic and Clinical Responses from Phase 1 / 2 Clinical Trials and Open-Label Continuous Dosing Sixteen patients (11 pediatric and 5 adult) were enrolled in Part 1 of 101A, and 15 patients advanced to Part 2 of 101A. Two patients discontinued the trial for personal reasons (one patient after Dose 3 of Part 1 and one patient after Dose 3 of Part 2). All 14 patients who completed Part 2 of 101A proceeded to the OLE trial.

[0188] The baseline characteristics of the patients are shown in Table 15. [Table 16]

[0189] Analysis of plasma arginine and guanidino compound levels revealed a marked and persistent decrease in plasma arginine levels (Figure 12(a) shows a 274 μM decrease from baseline at the median after 20 administrations of peginesatide. The decreases in plasma arginine from baseline to administration 1, administration 8, and OLE were statistically significant (p < 0.001). The decrease in plasma arginine was accompanied by a decrease in plasma guanidino compound (GC) levels. Figure 12(b) shows the plasma levels of guanidinoacetic acid (GAA), N-α-acetyl-L-arginine (NAA), α-keto-δ-guanidinovaleric acid (GVA), argininic acid (ARGA) at baseline and the decrease in plasma GC levels during OLE.

[0190] Fifteen out of 16 patients completed all mobility assessments at baseline (Patient 13 was wheelchair-bound) (Figure 13(a)). Deficits were defined as follows: 6MWT: less than the lower 5th percentile; GMFM part D: < 35 / 39; GMFM part E: < 68 / 72; ABAS-3: ≤ 85. Eighty-eight percent (14 / 16) of the patients had at least one motor deficit at baseline, and 88%, 50%, and 56% of the 16 patients were classified as having baseline deficits in 6MWT, GMFM part D, and GMFM part E, respectively. The adaptive behavior ABAS-3 assessment was available for 10 patients at baseline. Six patients did not undergo the test for technical reasons such as language, age, and cognitive impairment constraints. Eight out of 10 (80%) patients had baseline deficits in one or more domains evaluated by ABAS-3.

[0191] The overall clinical response, based on an improvement of ≥1 MCID in at least one of the 6MWT, GMFM-D, or GMFM-E assessments, showed that 11 out of 14 patients (79%) were defined as responders at dose 20 (Figure 13(b)). The 20-dose data showed that 6MWT, GMFM-D, and GMFM-E were sensitive enough to changes for the clinical benefit of ARG1-D patients to be achieved. The proportion of overall responders increased substantially from dose 8 to dose 20. All 5 patients (100%) who reached dose 44 maintained the overall clinical response status of responders at dose 20.

[0192] In all responders for individual components, ≥1 MCID was improved (Figure 13(b) and Figure 14). For 6MWT, 7 out of 13 patients (54%) were responders for this component only. The average change in 6MWT was 32 meters in all patients and 66 meters in 7 responders. For GMFM-D: 5 out of 8 patients (63%) with baseline deficits were responders for this component only (average MCID 1.84, range 1.21 - 3.33). For GMFM-E: 5 out of 8 patients (63%) with baseline deficits were responders for this component only (average MCID 4.79, range 1.67 - 8.33). The proportion of responders for individual mobility components was substantially greater at dose 20 compared to dose 8.

[0193] Data from all patients after 20 doses of pegzilarginase showed a marked and sustained decrease in plasma arginine, improvement in key disease symptoms, and a 79% clinical responder rate. The Phase 1 / 2 and OLE trials showed the value of using 6MWT, GMFM-D, or GMFM-E as tools for the clinical benefit of pegzilarginase to be achieved. Pegzilarginase was well tolerated and the incidence of treatment-related adverse events decreased over time. Evidence of improved arginine control and clinical benefit after pegzilarginase treatment provides further validation of the primary endpoints and design elements of the important Phase 3 PEACE trial (NCT03921541).

[0194] Example 20: Phase 3 Clinical Trial Design A randomized, double-blind, placebo-controlled Phase 3 trial regarding the efficacy and safety of Co-rhARG1-PEG in children and adults with arginase 1 deficiency is conducted using Co-rhARG1-PEG produced by the method of the present invention. This trial is currently ongoing as the Pegzilarginase Effect on Arginase 1 Deficiency Clinical Endpoints, or PEACE (CAEB1102-300A, NCT03921541).

[0195] The trial design of this Phase 3 trial is shown in the figure below: 25

Chemical formula

[0196] Main inclusion criteria a. Patients aged 2 years or older who are diagnosed with ARG1-D, have a plasma arginine level of ≧250 μmol / L, and for whom a statistical test of the proportion of patients achieving a plasma arginine of 200 μmol / L under medical guidance is possible b. Able to maintain a stable and consistent diet during the blinded period c. Able to stably administer ammonia scavengers, anti-epileptic therapy, and / or spasmolytic drugs during the blinded period d. Able to perform and successfully complete a clinical evaluation, and one of the components of the secondary clinical response evaluation items shown in Table 16 is missing at baseline

[0197] Main exclusion criteria a. Onset of hyperammonemia requiring hospitalization within 6 weeks before the start of treatment b. Active infectious diseases within 3 weeks before receiving the first pegzilarginase administration c. Extreme motor deficit (unable to walk at all) that cannot be evaluated by the Jilet Function Assessment Questionnaire (GFAQ) or is defined as a score of 1 on the GFAQ d. Having participated in previous interventional studies with peginesatide or currently participating in other clinical trials e. History of hypersensitivity to polyethylene glycol

Table 17

[0198] The primary endpoint of this Phase 3 trial is the reduction of plasma arginine (change from baseline in plasma arginine levels at week 24 based on treatment arm, change from baseline in individual patients in the active and placebo groups).

[0199] The secondary endpoint measures include the following: a. Clinical response assessment: Clinical responders are defined as patients who have improved in at least one of the 2MWT, GMFM-D, or GMFM-E clinical response assessments at week 24, as defined in Table 17 b. Efficacy rates of individual components of the clinical response assessment c. Other clinical outcome assessments i. Functional mobility assessment scales at 5, 50, and 500 meters ii. Jett Functional Ability Questionnaire (GFAQ) iii. Vineland Adaptive Behavior Scale II d. Safety assessment including immunogenicity e. Proportion of patients with plasma arginine < 200 μM within the normal range (40 - 115 μM) f. Characterization of the pharmacokinetic profile of peginesatide

Table 18

[0200] The total study period is expected to be approximately 178 weeks per subject, including a long-term open-label continuation period (3 - 4 weeks screening period, 24 weeks treatment period, followed by up to 150 weeks open-label continuation period). Subjects will receive Co-rhARG1-PEG or volume-adjusted placebo by IV infusion (approximately 30 minutes) once weekly. Dose changes of Co-rhARG1-PEG based on plasma arginine values alone will be performed by an open-label pharmacist and / or physician according to the dosing algorithm. After the first 8 weeks of the blinded long-term continuation period, subjects will have the option to receive Co-rhARG1-PEG by subcutaneous administration, subject to approval by the principal investigator and the sponsor. The initial mg / kg subcutaneous dose can be the same as the IV dose.

[0201] Subjects assigned to Co-rhARG1-PEG will start at dose level 2 (see Table 18 below), 0.10 mg / kg. From Visit 5 onwards, dose changes based on plasma arginine values, if necessary, will be performed by an open-label physician according to the following dosing algorithm: · If the plasma arginine level is >150 μM and the previous 2 doses of this sample are (a) at the same dose level in mg / kg and (b) consecutive (no missed doses), increase the dose by 2 dose levels (not exceeding 0.20 mg / kg) using a single 168-hour sample. · If the plasma arginine levels from two consecutive 168-hour samples (even if a dose was missed) are both <50 μM, decrease the dose by 1 dose level (see Table 17) and ensure it does not fall below 0.05 mg / kg.

Table 19

[0202] Statistical Considerations In the primary analysis, the mean decrease from baseline in plasma arginine levels of patients treated with pegvaliase and placebo will be compared 24 weeks after weekly dosing, based on the mean of the last 4 plasma arginine measurements that meet the strictly pre-specified criteria.

[0203] Specimen sizes of 10 and 20 patients randomized to placebo and pegargiminase, respectively, achieved 98% power to show 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 common SD of 120 μM.

[0204] Furthermore, this number of subjects provides > 80% power to detect a statistically significant difference of 40% between group ratios of clinical response endpoints at a significance level of 0.05 using Fisher's exact probability test.

[0205] Example 21: Site-Specific PEGylation Analysis An exemplary site-specific PEGylation analysis is shown in FIG. 15. Three Co-rhARG1-PEG batches were analyzed. Peptide mapping was performed by denaturing Co-rhARG1-PEG drug substance, Co-arginase 1 intermediate (non-PEGylated) lot and corresponding reference standards with guanidine-HCl, reducing with DTT, and alkylating with iodoacetamide. These samples were diluted in 50 mM Tris buffer pH 8.0. Each sample was digested with sequencing grade trypsin at 37° C. for approximately 5 hours. The resulting peptides were separated using a Waters Acquity BEH300C18 column, 2.1×150 mm, Waters C / N 186003687, with an acetonitrile gradient in 0.05% trifluoroacetic acid. LC-MS and MS / MS fragmentation of the peptides were obtained on a Waters Xevo G2-XS QTOF MS / MS.

[0206] As can be seen, none of the three batches were PEGylated at sites K3, K149, K190, K195, K29, K265, or K283. Also, PEGylation occurred at sites K16, K32, K38, K40, K47, K67, K74, K82, L87, K88, K152, K154, K171, K312, and K321 in all three batches. In some batches, the frequency of PEGylation was low at sites K222 and K223.

[0207] Throughout this specification, references 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 the embodiment are included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases "in one or more embodiments", "in certain embodiments", "in various embodiments", "in one embodiment", or "in an embodiment" in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments.

[0208] Although the disclosure of this specification has been provided with reference to certain embodiments, it should be understood that these embodiments are merely illustrative of the principles and applications of the present disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the present disclosure without departing from its spirit and scope. Accordingly, the present disclosure is intended to cover modifications and variations that are within the scope of the appended claims and their equivalents.

Claims

1. 1. A method for producing a purified recombinant cobalt-substituted human arginase, comprising: comprising an amino acid sequence that is at least 98% identical to SEQ ID NO:1, said method comprising: a. culturing E. coli cells producing rhARG in a bioreactor; b. Lysing the E. Coli cells; c. Removing cell debris from the lysate; d. loading the cell lysate onto a cation exchange column; e. Eluting the rhARG with a high salt solution; f. incubating the eluted rhARG with a cobalt salt to form cobalt-substituted rhARG (Co-rhARG); g. applying the Co-rhARG to an anion exchange column and collecting the flow-through; h. applying the flow-through to a third chromatography column; i. eluting the Co-rhARG from the third chromatographic column with a high salt solution.

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

3. 3. The method of claim 1 or 2, wherein eluting the rhARG from the cation exchange column uses a high salt solution having a salt concentration of up to about 0.5 M.

4. 3. The method of claim 1 or 2, wherein eluting the rhARG from the cation exchange column uses a high salt solution having a salt concentration of about 0.1 M.

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

6. 3. The method of claim 1 or 2, wherein eluting the rhARG from the cation exchange column uses a salt gradient of about 0.0 to about 0.2M.

7. 7. The method of any one of claims 1 to 6, wherein the cobalt salt comprises a Co2+ salt.

8. 8. The method of claim 1, wherein the cobalt salt comprises CoCl2.

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

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

11. The PEGylated protein is selected from the group consisting of K16, K32, K38, K40, K47, K67, K74, K82, L87, 11. The method of claim 10, comprising one or more of the PEGylated amino acid residues at K88, K152, K154, K171, K222, K223, K312 and K321.

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

13. The PEGylated protein is 13. The method of any one of claims 10 to 12, comprising PEGylated amino acid residues at least at K16, K32, K38, K40, K47, K67, K74, K82, L87, K88, K152, K154, K171, K312 and K321.

14. 14. The method of any one of claims 10 to 13, wherein the pegylated protein has no pegylated amino acid residues at K3, K149, K190, K195, K29, K265 and K283.

15. 1. A method for producing purified PEGylated recombinant cobalt-substituted human arginase, comprising: The recombinant human arginase (rhARG) comprises an amino acid sequence that is at least 98% identical to SEQ ID NO:1, and the method comprises: a. culturing E. coli producing rhARG in a bioreactor; b. Lysing the E. Coli cells; c. Removing cell debris from the lysate; d. loading the cell lysate onto a cation exchange column; e. Eluting the rhARG with a high salt solution; f. incubating the eluted rhARG with 10 mM CoCl2 to form cobalt-substituted rhARG (Co-rhARG); g. applying the Co-rhARG to an anion exchange column and collecting the flow-through; h. applying the flow-through to a multimodal chromatography (MMC) column; i. eluting the Co-rhARG from the MMC column with a high salt solution; j. adding a molar excess of methoxy PEG succinimidyl carboxymethyl ester; k. removing excess PEG.

16. 1. A method for producing a purified recombinant cobalt-substituted human arginase, comprising: comprising an amino acid sequence that is at least 98% identical to SEQ ID NO:1, said method comprising: a. culturing rhARG producing E. coli cells in a bioreactor at about 36° C. to about 38° C. and a pH of about 7.0 to about 7.4 with agitation and aeration; i. Adjust the temperature of the bioreactor to about 29° C.; ii. Inducing E. Coli cells to produce rhARG; iii. Cultivating the E. coli cells for about 18 hours; iv. harvesting the E. coli cells by centrifugation; culturing; b. lysing the E. coli cells by high pressure homogenization in 25 mM HEPES at a pH between about 7.2 and about 7.6 and at or below about 15° C.; c. removing cellular debris from the lysate by centrifugation at or below 15° C. and filtering the lysate through a 0.8 micron filter followed by a 0.5 micron filter; d. loading the cell lysate onto a cation exchange column and then washing the column with 25 mM HEPES, pH 7.2-7.6; e. Eluting the rhARG with a high salt solution containing 25 mM HEPES, 0.1 M NaCl, pH 7.2-7.6 at room temperature; f. incubating the eluted rhARG with 10 mM CoCl2 at room temperature for about 2 to about 8 hours to form cobalt-substituted rhARG (Co-rhARG); i. exchanging Co-rhARG with 50 mM Tris, pH 8.1-8.5; g. applying the Co-rhARG to an anion exchange column and collecting the flow-through; h. applying the flow-through to a Capto Multimodal Chromatography (MMC) column; i. eluting the Co-rhARG from the MMC column with a high salt solution comprising 50 mM Tris, 250 mM NaCl, pH 8.1-8.

5.

17. 1. A method for producing a purified recombinant PEGylated recombinant cobalt-substituted human arginase, comprising: comprising an amino acid sequence that is at least 98% identical to SEQ ID NO:1, said method comprising: a. culturing rhARG producing E. coli in a bioreactor at about 36° C. and about 38° C., at a pH of about 7.0 to about 7.4, with agitation and aeration; i. Adjust the temperature of the bioreactor to about 29° C.; ii. Inducing E. coli to produce rhARG; iii. Cultivating the E. coli for about 18 hours; iv. harvesting the E. coli cells by centrifugation; culturing; b. The E. coli cells are homogenized by high pressure homogenization in 25 mM HEPES at a pH between about 7.2 and about 7.6 and at about 15° C. or less. Dissolving and c. removing cellular debris from the lysate by centrifugation at or below 15° C. and filtering the lysate through a 0.8 micron filter followed by a 0.5 micron filter; d. Loading the cell lysate onto a cation exchange column and then Washing with 25 mM HEPES, pH 7.2-7.6; e. Eluting the rhARG with a high salt solution containing 25 mM HEPES, 0.1 M NaCl, pH 7.2-7.6 at room temperature; f. incubating the eluted rhARG with 10 mM CoCl2 at room temperature for about 2 to about 8 hours to form cobalt-substituted rhARG (Co-rhARG); i. exchanging Co-rhARG with 50 mM Tris, pH 8.1-8.5; g. applying the Co-rhARG to an anion exchange column and collecting the flow-through; h. applying the flow-through to a Capto Multimodal Chromatography (MMC) column; i. eluting the Co-rhARG from the MMC column with a high salt solution comprising 50 mM Tris, 250 mM NaCl, pH 8.1-8.5 (MMC buffer); i. Replace the MMC buffer with 20 mM sodium phosphate, 50 mM NaCl, 1.5% glycerol, pH 7.4 (Buffer 1) to adjust the protein concentration to approximately 5.0 mg / mL; ii. Eluting by exchanging Buffer 1 with 0.1 M sodium phosphate, pH 8.1-8.5 (Buffer 2) to concentrate the protein concentration to about 10.0 mg / mL; j. A molar excess of methoxy PEG succinimidyl carboxymethyl ester, approximate molecular weight 5,000 Da, is added (about 19 moles of methoxy PEG succinimidyl carboxymethyl ester is added per mole of protein) and the mixture is stirred for about 30 minutes to about 4 hours. Incubating at pH 8.4; k. Replacing Buffer 2 with 20 mM sodium phosphate, 50 mM NaCl, 1.5% glycerol, pH 7.4 to remove excess PEG.

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

19. 19. The composition of claim 18, wherein the protein is covalently linked to polyethylene glycol at one or more of K16, K32, K38, K40, K47, K67, K74, K82, L87, K88, K152, K154, K171, K222, K223, K312 and K321.

20. A composition comprising a recombinant human arginase (rhARG) protein, said protein having an amino acid sequence that is at least 98% identical to SEQ ID NO:

1. wherein the protein is complexed with a non-native metal cofactor, the non-native metal cofactor being cobalt, and the protein is covalently attached to polyethylene glycol at one or more of K16, K32, K38, K40, K47, K67, K74, K82, L87, K88, K152, K154, K171, K222, K223, K312 and K321.

21. The recombinant human arginase (rhARG) 21. The composition of any one of claims 18 to 20, comprising 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.

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

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

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

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

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

27. The exogenous protein fragment comprises an Fc region of an immunoglobulin or a portion of an Fc region of an immunoglobulin.

27. The composition of claim 26.

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

29. the protein exhibits a kcat / Km for hydrolysis of arginine in the range of about 200 mM-1s-1 to about 4,000 mM-1s-1 at pH 7.4 when assayed in vitro; The composition according to any one of claims 18 to 28.

30. 30. The method of claim 29, wherein the protein exhibits a kcat / KM for the hydrolysis of arginine in the range of about 400 mM-1 s-1 to about 2,500 mM-1 s-1 at pH 7.4 when assayed in vitro.

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

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

33. 33. The composition of any one of claims 18 to 32, wherein the total cobalt content of the composition ranges from about 9 μg / mL to about 15 μg / mL.

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

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

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

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

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

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

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

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

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

43. Administering the pharmaceutical composition comprises:

43. The method of any one of claims 40 to 42, starting at a dose of 0.1 mg / kg based on the weight of the non-pegylated enzyme.

44. 44. The method of any one of claims 40 to 43, further comprising monitoring plasma arginine levels in the patient.

45. The dose is It is prepared according to the following algorithm: a. If the plasma arginine level is >150 μM, then use a single 168 hour sample to increase the dose by 2 dose levels in the table below (not to exceed 0.20 mg / kg) if the two doses prior to this sample were a) the same dose level in mg / kg and b) consecutive (no missed dose). b. The method of any one of claims 40-44, wherein if the plasma arginine levels from two consecutive 168 hour samples (even if a missed dose) are both <50 μM, then the dose is reduced by one dose level in the table below, but not below 0.05 mg / kg. 【Table 1】

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