Process for manufacture of nematode-extracted anticoagulant protein (NAP)

Abstract

The present invention provides a process for manufacture of purified Nematode-extracted Anticoagulant Proteins (NAPs), wherein the NAP manufactured by the claimed process method is a NAP drug substance that can be formulated as a NAP drug product. The present invention provides NAP drug substances and NAP drug products manufactured by the process disclosed herein. In one embodiment, the present invention provides a process for manufacture of rNAPc2/proline drug substance and rNAPc2/proline drug product, and provides rNAPc2/proline drug substance manufactured by the process disclosed herein.

1 . A process for manufacture of nematode-extracted anticoagulant protein (NAP) drug substance comprising:
 (a) a fermentation process comprising producing NAP in a suitable host, wherein at least one sequence encoding NAP is integrated into the genome of the host;   (b) a recovery process comprising separating NAP from cells and cellular debris; and   (c) a purification process comprising purifying NAP drug substance away from contaminants.   
     
     
         2 . The process of  claim 1 , wherein the purification process further comprises introducing NAP substance into final drug formulation. 
     
     
         3 . The process of  claim 2 , further comprising a fill process to produce NAP drug product. 
     
     
         4 . The process of  claim 1 , wherein the NAP is selected from the group consisting of rNAPc2, rNAPc2/proline, AcaNAP5, AcaNAP6, AcaNAP23, AcaNAP31, AcaNAP42, AcaNAP48, AceNAP5, AceNAP7, AduNAP4, AcaNAP24, AcaNAP25, AcaNAP44, or AcaNAP46. 
     
     
         5 . The process of  claim 1 , wherein the NAP is rNAPc2/proline. 
     
     
         6 . The process of  claim 2 , wherein the NAP is selected from the group consisting of rNAPc2 (AcaNAPc2), rNAPc2/proline (AcaNAPc2/proline), AcaNAP5, AcaNAP6, AcaNAP23, AcaNAP31, AcaNAP42, AcaNAP48, AceNAP5, AceNAP7, AduNAP4, AcaNAP24, AcaNAP25,AcaNAP44, or AcaNAP46. 
     
     
         7 . The process of  claim 2 , wherein the NAP is rNAPc2/proline. 
     
     
         8 . The process of  claim 1 , wherein the host is  Pichia pastoris    
     
     
         9 . The process of  claim 1 , wherein the fermentation process comprises a seed fermentation process wherein host cells are grown to a desired cell density and a production fermentation process wherein NAP is produced to a desired titer. 
     
     
         10 . The process of  claim 9 , wherein the production fermentation process comprises glycerol batch fermentation, glycerol fed-batch fermentation, methanol adaptation fermentation, and methanol induction fermentation. 
     
     
         11 . The process of  claim 10 , further comprising controlling the pH range for fermentation at about 2.9±0.1 pH units during the methanol adaptation fermentation and the methanol induction fermentation. 
     
     
         12 . The process of  claim 10 , further comprising controlling temperature for fermentation. 
     
     
         13 . The process of  claim 12 , comprising maintaining the temperature of the methanol adaptation phase of the fermentation at about 28±2° C. for about the first four hours and at about 25±1° C. for the remainder of the methanol adaptation phase. 
     
     
         14 . The process of  claim 10 , wherein production fermentation is carried out for up to about seven days, during which period the NAP titer continues to increase. 
     
     
         15 . The process of  claim 1 , wherein the recovery process comprises expanded-bed ion-exchange chromatography. 
     
     
         16 . The process of  claim 15 , comprising Streamline SP XL ion exchange resin expanded bed chromatography. 
     
     
         17 . The process of  claim 1 , wherein the purification process comprises hydrophobic interaction chromatography, collecting NAP fractions, at least one ultrafiltration/diafiltration (UF/DF) of NAP fractions, ion exchange chromatography, and collecting NAP fractions from ion-exchange chromatography. 
     
     
         18 . The process of  claim 17 , wherein the NAP fractions from ion-exchange chromatography contain NAP drug substance. 
     
     
         19 . The process of  claim 17 , wherein hydrophobic interaction chromatography comprises utilizing Source 15PHE hydrophobic interaction chromatography media at about pH3.0±0.1 
     
     
         20 . The process of  claim 17 , wherein ion exchange chromatography comprises utilizing Source 15Q ion chromatography media. 
     
     
         21 . The process of  claim 17 , further comprising at least one final UF/DF of fractions from ion-exchange chromatography. 
     
     
         22 . The process of  claim 21 , wherein the UF/DF exchanges NAP drug substance into final drug formulation buffer to generate NAP drug product. 
     
     
         23 . The process of  claim 3 , wherein the fill process comprises bulk filtration of NAP drug substance in final drug formulation. 
     
     
         24 . The process of  claim 23 , further comprising a fill step of dispensing NAP drug substance in dosage form to produce NAP drug product. 
     
     
         25 . The process of  claim 24 , wherein the fill process further comprises lyophilization of NAP drug product. 
     
     
         26 . A process for manufacture of rNAPc2/proline drug substance comprising:
 (a) a fermentation process wherein rNAPc2/proline is produced in  Pichia pastoris  having at least one sequence encoding rNAPc2/proline is integrated into the genome, comprising a seed fermentation to grow host cells to a desired cell density and a production fermentation process comprising glycerol batch fermentation, glycerol fed-batch fermentation, methanol adaptation fermentation, and methanol induction fermentation, for up to about seven days;   (b) a recovery process comprising ion exchange expanded bed chromatography to separate rNAPc2/proline from cells and cellular debris; and   (c) a purification process comprising hydrophobic interaction chromatography utilizing hydrophobic interaction chromatography media, collecting rNAPc2/proline fractions, at least one ultrafiltration/diafiltration (UF/DF) of rNAPc2/proline fractions, ion exchange chromatography, and collecting rNAPc2/proline fractions from ion-exchange chromatography;   wherein the rNAPc2/proline fractions from ion-exchange chromatography contain rNAPc2/proline drug substance.   
     
     
         27 . The process of  claim 26 , further comprising controlling temperature for fermentation. 
     
     
         28 . The process of  claim 27 , comprising maintaining the temperature of the methanol adaptation fermentation at about 28±2° C. for about the first four hours and at about 25±1° C. for the remainder of the methanol adaptation fermentation. 
     
     
         29 . The process of  claim 26 , comprising maintaining the pH at about 2.9±0.1 during the methanol adaptation fermentation and the methanol induction fermentation. 
     
     
         30 . The process of  claim 26 , comprising Streamline SP XL ion exchange resin expanded bed chromatography at a pH of about 3.2±0.2. 
     
     
         31 . The process of  claim 26 , comprising Source 15PHE hydrophobic interaction chromatography at about pH 3.0±0.1. 
     
     
         32 . The process of  claim 26 , comprising Source 15Q ion chromatography. 
     
     
         33 . The process of  claim 26 , further comprising at least one final UF/DF of fractions from ion-exchange chromatography. 
     
     
         34 . A process for manufacture of rNAPc2/proline liquid drug product comprising the process of  claim 26  and further comprising introducing rNAPc2/proline drug substance into final drug formulation, a fill process comprising bulk filtration of rNAPc2/proline drug substance in final drug formulation, and a fill step comprising dispensing rNAPc2/proline in dosage form into a container to generate rNAPc2/proline liquid drug product. 
     
     
         35 . A process for manufacture of rNAPc2/proline lyophilized drug product comprising the process of  claim 34  and further comprising lyophilization of the rNAPc2/proline liquid drug product in the container. 
     
     
         36 . A NAP drug substance manufactured by the process of  claim 1 . 
     
     
         37 . A NAP drug substance of  claim 36 , wherein NAP is selected from rNAPc2 (AcaNAPc2), rNAPc2/proline (AcaNAPc2/proline), AcaNAP5, AcaNAP6, AcaNAP23, AcaNAP31, AcaNAP42, AcaNAP48, AceNAP5, AceNAP7, AduNAP4, AcaNAP24, AcaNAP25, AcaNAP44, or AcaNAP46. 
     
     
         38 . A NAP drug substance of  claim 37 , wherein NAP is rNAPc2/proline. 
     
     
         39 . A NAP drug product manufactured by the process of  claim 2 . 
     
     
         40 . A NAP drug product of  claim 39 , wherein NAP is selected from rNAPc2 (AcaNAPc2), rNAPc2/proline (AcaNAPc2/proline), AcaNAP5, AcaNAP6, AcaNAP23, AcaNAP31, AcaNAP42, AcaNAP48, AceNAP5, AceNAP7, AduNAP4 AcaNAP24, AcaNAP25, AcaNAP44, or AcaNAP46. 
     
     
         41 . A NAP drug product of  claim 40 , wherein NAP is rNAPc2/proline. 
     
     
         42 . A rNAPc2/proline drug substance of  claim 26 . 
     
     
         43 . A rNAPc2/proline liquid drug product of  claim 34 . 
     
     
         44 . A rNAPc2/proline lyophilized drug product of  claim 35 .