C3 fusion protein and methods of making and using thereof

Abstract

The present invention provides, among other things, improved therapeutic compositions comprising a C3 fusion protein and methods of making and using the same. In particular, the present invention provides improved methods for the treatment of spinal cord injury and other CNS trauma and/or facilitate axon growth or other tissue repair.

1 . A method for producing recombinant ADP-ribosyl transferase C3 (C3 fusion protein), comprising: cultivating host cells comprising a nucleic acid encoding a recombinant C3 fusion protein having an amino acid sequence of SEQ ID NO:2 in a large scale vessel under conditions that:
 promote expression of the C3 fusion protein at a titer concentration of greater than 1 g/L; and/or   involve a fermentation process to promote expression of the C3 fusion protein.   
     
     
         2 . The method of  claim 1 , wherein the fermentation process is a fed batch culturing process. 
     
     
         3 . The method of  claim 2 , wherein:
 the fed batch culturing process includes a batch mode, a first stage of exponential feeding, and a second stage of constant feeding;   the carbon source is selected from glycerol, glucose, sucrose, lactose, arabinose, maltotriose, sorbitol, xylose, rhamnose, and/or mannose; and/or   the method further comprises a step of adding an inducing agent to trigger expression of the C3 fusion protein.   
     
     
         4 . The method of  claim 3 , wherein:
 the batch mode lasts for at least about 7 hours, the first stage of exponential feeding lasts for about 7-8 hours and the second stage of constant feeding lasts for about 8 hours;   the second stage of constant feeding is at a feed rate at the end of the exponential feeding stage;   the first exponential feeding stage is maintained at a temperature between 34° C. to 40° C., and wherein the second and/or third constant feeding stage is maintained at a temperature between 24° C. to 32° C.; and/or   the method further comprises a third stage of constant feeding.   
     
     
         5 . The method of  claim 4 , wherein:
 the feed rate at the third stage of constant feeding is the same as the second stage; and/or   the third stage of constant feeding lasts for at least about 4 hours.   
     
     
         6 . The method of  claim 3 , wherein the carbon source at the first stage is a first sugar and the carbon source at the second stage is a second sugar;
 wherein the first sugar and the second sugar are different sugars or wherein the first sugar and the second sugar are identical sugars.   
     
     
         7 . The method of  claim 6 , wherein the first sugar is selected from the group consisting of glycerol, glucose, sucrose, and any combination thereof and the second sugar is selected from the group consisting of glycerol, glucose, sucrose, and any combination thereof; or wherein the identical sugars comprise glycerol. 
     
     
         8 . The method of  claim 1 , wherein:
 the large scale vessel is a bioreactor;   the host cells are bacterial cells; and/or   the method further comprises a step of recovering the expressed C3 fusion protein from the host cells.   
     
     
         9 . The method of  claim 8 , wherein:
 the bacterial cells are  E. coli ; and/or   the method further comprises purifying the expressed recombinant C3 fusion protein.   
     
     
         10 . The method of  claim 9 , wherein at least 80%, 85%, 90%, or 95% of the purified recombinant C3 fusion protein recovered from the host cells do not contain a methionine at the N-terminus. 
     
     
         11 . The method of  claim 1 , further comprising purifying recombinant ADP-ribosyl transferase C3 (C3 fusion protein), comprising: providing a biological sample comprising a recombinant C3 fusion protein having an amino acid sequence of SEQ ID NO: 1, and subjecting the biological sample to a series of chromatography steps comprising at least one cation exchange chromatography step and at least one hydrophobic interaction chromatography step. 
     
     
         12 . A method of purifying recombinant ADP-ribosyl transferase C3 (C3 fusion protein), comprising: providing a biological sample comprising a recombinant C3 fusion protein having an amino acid sequence of SEQ ID NO: 1, and subjecting the biological sample to a series of chromatography steps comprising at least one cation exchange chromatography step and at least one hydrophobic interaction chromatography step. 
     
     
         13 . The method of  claim 12 , wherein:
 the at least one cation exchange chromatography step includes two or more cation exchange chromatography steps;   the at least one hydrophobic interaction chromatography step follows the at least cation exchange chromatography step; and/or   the series of chromatography steps comprise a first cation exchange chromatography step, a second cation exchange chromatography step and a hydrophobic interaction chromatography step, in that order.   
     
     
         14 . The method of  claim 12 , wherein:
 the at least one cation exchange chromatography step uses a column comprising SP Sepharose;   the at least one cation exchange chromatography step uses a column with a bed height of 5 cm-30 cm;   the at least one cation exchange chromatography step uses a column with a loading velocity of 100 cm/hr-200 cm/hr;   the at least one cation exchange chromatography step uses a column with a load conductivity of 7 mS/cm-8 mS/cm;   the at least one hydrophobic interaction chromatography step uses a column comprising Butyl 650M resin;   the at least one hydrophobic interaction chromatography step uses a column with a bed height of 10 cm-30 cm;   the method further comprises one or more steps of Ultrafiltration/Diafiltration I (UF/DF I) and membrane adsorption before, between or after the chromatography steps;   the method further comprises a step of concentrating the purified recombinant C3 fusion protein to a concentration of 0.1 mg/mL-40 mg/mL; and/or   the biological sample comprises a homogenate of cells expressing the recombinant C3 fusion protein.   
     
     
         15 . The method of  claim 14 , wherein the cells are bacterial cells; and/or
 wherein the cells comprise a nucleic acid encoding a recombinant C3 fusion protein having an amino acid sequence of SEQ ID NO: 2.   
     
     
         16 . The method of  claim 15 , wherein at least 80% of the purified recombinant C3 fusion protein does not contain a methionine at the N-terminus, or wherein substantially all purified recombinant C3 fusion proteins do not contain a methionine at the N-terminus. 
     
     
         17 . The method of  claim 15 , wherein at least 85% of the purified recombinant C3 fusion protein does not contain a methionine at the N-terminus, or wherein substantially all purified recombinant C3 fusion proteins do not contain a methionine at the N-terminus. 
     
     
         18 . The method of  claim 15 , wherein at least 95% of the purified recombinant C3 fusion protein does not contain a methionine at the N-terminus, or wherein substantially all purified recombinant C3 fusion proteins do not contain a methionine at the N-terminus. 
     
     
         19 . The method of  claim 16 , wherein:
 the purified recombinant C3 fusion protein has a purity of equal to or greater than 85% measured by main peak of IE-HPLC;   the purified recombinant C3 fusion protein has a purity equal to or greater than 90% measured by the main peak of IE-HPLC; and/or   the purified recombinant C3 fusion protein has a concentration of about 31-37 mg/mL.   
     
     
         20 . A C3 recombinant ADP-ribosyl transferase protein composition purified according to a method of  claim 12 .