Harvest operations for recombinant proteins

Abstract

The present invention contemplates a method of producing a recombinant protein comprising (a) fermenting a prokaryotic host cell wherein said prokaryotic host cell has been transformed with a nucleic acid encoding said recombinant protein, and (b) harvesting said recombinant protein under conditions where dO2 levels are greater than 0%, and (c) purifying said recombinant protein to a filtered bulk, wherein said filtered bulk does not contain detectable DHNA-recombinant protein adduct, as measured by an IEC assay at 310 nm. The present invention also contemplates a method of producing a recombinant protein comprising (a) fermenting a menE gene-deleted prokaryotic host cell wherein said prokaryotic host cell has been transformed with a nucleic acid encoding said recombinant protein, (b) harvesting said recombinant protein; and (c) purifying said recombinant protein to a filtered bulk, wherein said filtered bulk does not contain detectable DHNA-recombinant protein adduct, as measured by an IEC assay at 310 nm, and wherein the recombinant protein yield is increased by about 20% or greater.

1 . A method of producing a recombinant protein comprising (a) fermenting a prokaryotic host cell wherein said prokaryotic host cell has been transformed with a nucleic acid encoding said recombinant protein, and (b) harvesting said recombinant protein under conditions where dissolved oxygen (dO 2 ) levels are greater than 0%, and (c) purifying said recombinant protein to a filtered bulk for storage (FBS), wherein said filtered bulk does not contain detectable 1,4-dihydroxy-2-naphthoate (DHNA)-recombinant protein adduct, as measured by an ion exchange chromatography (IEC) assay at 310 nm. 
     
     
         2 . The method of  claim 1 , wherein said analytical assay is by HPLC, RP HPLC, HIC HPLC, NMR, mass spectrometry, or UV spectroscopy. 
     
     
         3 . The method of  claim 1 , wherein said dO 2  is maintained at levels greater than 0% continuously throughout the harvest operations of step (b). 
     
     
         4 . The method of  claim 3 , wherein the harvest operations comprise a homogenization stage. 
     
     
         5 . The method of  claim 4 , wherein said dO 2  is maintained at about 30% to about 75% prior to homogenization. 
     
     
         6 . The method of  claim 4 , wherein said dO 2  is maintained at levels greater than 75% prior to homogenization. 
     
     
         7 . The method of  claim 4  or  5 , wherein said dO 2  is maintained at about 50% after homogenization. 
     
     
         8 . The method of  claim 4 , wherein said dO 2  is maintained at levels greater than 50% after homogenization. 
     
     
         9 . The method of  claim 5 , wherein said dO 2  is maintained for a period of greater than or equal to 1.5 hours. 
     
     
         10 . (canceled) 
     
     
         11 . The method of  claim 1 , wherein said dO 2  is maintained with overlay or sparged air, with increased back-pressure, or with agitation rate. 
     
     
         12 . The method of  claim 11 , wherein the overlay air is from about 0.4 vvm to about 0.8 vvm. 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 11 , wherein the increased backpressure is between about 1.0 to 30 psi. 
     
     
         15 . The method of  claim 14 , wherein the increase backpressure is targeted at 19 psi. 
     
     
         16 . The method of  claim 11 , wherein the agitation rate is from about 6 Watts/L to about 8 Watts/L. 
     
     
         17 . (canceled) 
     
     
         18 . A method of producing a recombinant protein comprising (a) fermenting a  menE  gene-deleted prokaryotic host cell wherein said prokaryotic host cell has been transformed with a nucleic acid encoding said recombinant protein, (b) harvesting said recombinant protein; and (c) purifying said recombinant protein to a FBS, wherein said filtered bulk does not contain detectable 1,4-dihydroxy-2-naphthoate (DHNA)-recombinant protein adduct, as measured by an ion exchange chromatography (IEC) assay at 310 nm. 
     
     
         19 . (canceled) 
     
     
         20 . (canceled) 
     
     
         21 . The method of  claim 1 , wherein said recombinant protein is a recombinant polypeptide or an isolated antibody. 
     
     
         22 . The method of  claim 1 , wherein said prokaryotic host cell is  Escherichia coli (E. coli), Enterobacter, Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsiella, Proteus, Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus.   
     
     
         23 . A method of generating a menE gene-deleted  E.   coli  host cell comprising 
 transforming an  E.   coli  cell with a PCR product comprising a resistance cassette for kanamycin flanked by FLP recognition target sites and a 50 base pair homology to chromosomal sequences adjacent to the menE gene and selecting for kanamycin resistance, and thereby generating a menE gene deleted  E.   coli  cell. 
 
     
     
         24 . A prokaryotic host cell comprising a deletion in the menE gene and a nucleic acid encoding a recombinant protein.