Methods For Production Of Human Recombinant Arginase 1 And Uses Thereof

Abstract

Described are methods for producing recombinant Arginase, such as PEGylated, cobalt-substituted recombinant human Arginase 1. Also described are pharmaceutical compositions comprising such recombinant Arginase, as well as methods of treatment and uses of such recombinant Arginase.

1 . A method for producing purified recombinant cobalt-substituted human Arginase, wherein the recombinant human Arginase (rhARG) comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1, the method comprising:
 a. in a bioreactor, culturing  E. coli  cells that produce rhARG;   b. lysing the  E. coli  cells;   c. removing cell debris from the lysate;   d. loading the cell lysate on 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 said Co-rhARG to an anion exchange column and collecting the flow-through;   h. applying the flow-through onto a third chromatography column; and   i. eluting the Co-rhARG from the third chromatography column with a high salt solution.   
     
     
         2 . The method of  claim 1 , wherein up to 60 grams of rhARG is loaded onto the cation exchange column per liter of cation exchange resin. 
     
     
         3 . The method of  claim 1 , wherein eluting the rhARG from the cation exchange column uses a high salt solution of up to about 0.5 M salt concentration. 
     
     
         4 . The method of  claim 1 , wherein eluting the rhARG from the cation exchange column uses a high salt solution of about 0.1 M salt concentration. 
     
     
         5 . The method of  claim 1 , wherein eluting the rhARG from the cation exchange column uses a gradient of about 0.0 to about 0.5 M salt concentration. 
     
     
         6 . The method of  claim 1 , wherein eluting the rhARG from the cation exchange column uses a gradient of about 0.0 to about 0.2M salt concentration. 
     
     
         7 . The method of  claim 1 , wherein the cobalt salt comprises a Co 2+  salt. 
     
     
         8 . The method of  claim 1 , wherein the cobalt salt comprises CoCl 2 . 
     
     
         9 . The method of  claim 1 , wherein the third chromatography column comprises a multimodal chromatography (MMC) column. 
     
     
         10 . The method of  claim 1 , further comprising reacting the rhARG or Co-rhARG with a PEGylation reactant to provide a PEGylated protein. 
     
     
         11 . The method of  claim 10 , wherein the PEGylated protein comprises one or more of PEGylated amino acid residues at K16, K32, K38, K40, K47, K67, K74, K82, L87, K88, K152, K154, K171, K222, K223, K312 and K321. 
     
     
         12 . The method of  claim 11 , wherein the PEGylated protein comprises one of 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 is PEGylated. 
     
     
         13 . The method of  claim 10 , wherein 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. 
     
     
         14 . The method of  claim 13 , wherein the PEGylated protein does not have PEGylated amino acid residues at K3, K149, K190, K195, K29, K265 and K283. 
     
     
         15 . (canceled) 
     
     
         16 . (canceled) 
     
     
         17 . A method for producing purified recombinant PEGylated recombinant cobalt-substituted human Arginase, wherein the recombinant human Arginase (rhARG) comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1, the method comprising:
 a. in a bioreactor, culturing  E. coli  that produce rhARG at about 36° C. and about 38° C., and pH of between about 7.0 and about 7.4, with agitation and aeration;
 i. adjusting the temperature of the bioreactor to about 29° C.; 
 ii. inducing  E. coli  to produce for rhARG; 
 iii. culturing the  E. coli  for about 18 hours; 
 iv. harvesting the  E. coli  by centrifugation; 
   b. lysing the  E. coli  cells by high pressure homogenization in 25 mM HEPES at a pH of about 7.2 to about 7.6, and at or below about 15° C.;   c. removing cell debris from the lysate by centrifugation at or below 15° C., then filtering the lysate with an about 0.8 micron filter then filtering with an about 0.5 micron filter;   d. loading the cell lysate on a cation exchange column then washing the column with 25 mM HEPES, pH 7.2-7.6;   e. eluting the rhARG at room temperature with a high salt solution comprising 25 mM HEPES, 0.1M NaCl, pH 7.2-7.6;   f. incubating the eluted rhARG with 10 mM CoCl 2  at room temperature for about 2 to about 8 hours to form cobalt-substituted rhARG (Co-rhARG);
 i. exchanging Co-rhARG into 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 onto 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. exchanging the MMC Buffer for 20 mM Sodium Phosphate, 50 mM NaCl, 1.5% Glycerol, pH 7.4 (Buffer 1), and adjusting the concentration of protein to about 5.0 mg/mL; 
 ii. exchanging Buffer 1 for 0.1M sodium phosphate, pH 8.1-8.5 (Buffer 2), and concentrating the protein concentration to about 10.0 mg/mL; 
   j. adding a molar excess of methoxy PEG succinimidyl carboxymethyl ester 5,000 Da approximate molecular weight, wherein about 19 moles of methoxy PEG succinimidyl carboxymethyl ester is added for each mole of protein and incubating this mixture for about 30 minutes to about 4 hours at approximately pH 8.4;   k. removing excess PEG by exchanging Buffer 2 for 20 mM Sodium Phosphate, 50 mM NaCl, 1.5% Glycerol, pH 7.4.   
     
     
         18 . A composition comprising Co-rhARG or Co-rhARG-PEG produced by the method of  claim 1 . 
     
     
         19 . The composition of  claim 18 , wherein the protein is covalently linked to a 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, wherein the protein comprises an amino acid sequence that is at least 98% identical to SEQ ID NO: 1, wherein the protein is in a complex with a non-native metal cofactor, wherein the non-native metal cofactor is cobalt, and wherein the protein is covalently linked to a 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 - 33 . (canceled) 
     
     
         34 . The composition of  claim 20 , wherein the composition produces at least 9 peaks when loaded on imaging capillary isoelectric focusing (iCIEF), wherein peak 1 is less than 20%, peak 2 is less than 30%, Peak 3+4 is in the range of 10-30%, peak 5 is in the range of 15-30%, peak 6 is in the range of 10-25%, peak 7 is less than 25%, peak 8 is less than 15%, and peak 9 is less than 8%. 
     
     
         35 . The composition of  claim 20 , wherein the composition produces at least 9 peaks when loaded on icIEF, wherein peak 1 is in the range of 5-7%, peak 2 is 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%. 
     
     
         36 - 45 . (canceled)