Fed-batch fermentation process and culture medium for the production of plasmid DNA in E. coli on a manufacturing scale

Abstract

A process for producing plasmid DNA E. coli cells comprises a pre-culture and fed-batch process. The culture media of the batch phase and the culture medium added during the feeding phase are chemically defined. The culture medium of the feeding phase contains a growth-limiting substrate and is added, for at least a fraction of the feeding phase, at a feeding rate that follows a pre-defined exponential function, thereby controlling the specific growth rate at a pre-defined value. The process results in high yield and homogeneity of plasmid DNA.

1 . A process for producing plasmid DNA on a manufacturing scale, said method comprised of the steps of: 
 a) growing  E. coli  cells that bear a plasmid carrying a gene of interest in a pre-culture and subsequently fermenting in a main culture;    b) recovering and purifying the plasmid DNA from said main culture;    wherein said main culture is a fed-batch process comprising a batch phase and a feeding phase and    the culture medium of said batch phase and feeding phase being chemically defined and    the culture medium of said feeding phase further comprised of a growth-limiting substrate and said culture medium being added at a feeding rate that follows, for at least a fraction of the feeding phase, a pre-defined exponential function, wherein said exponential function is      F t =const*e μt , wherein  F t  is the flow rate [L/h] of the feed medium;    μ is the specific growth rate [h −1 ], and    t is the time interval [h] from start of the feeding phase.      
   
   
       2 . The method of  claim 1 , wherein said fraction of said feeding phase during which said feeding rate follows said exponential function is such that more than about 20% of the total dry cell weight to be obtained in the feeding phase is generated during said fraction.  
   
   
       3 . The process of  claim 1 , wherein said preculture medium is chemically defined.  
   
   
       4 . The process of  claim 1 , using  E. Coli  cells with a mutation in the relA gene.  
   
   
       5 . The process of  claim 4 , wherein the  E. coli  strain is K-12.  
   
   
       6 . The process of  claim 5 , wherein the  E. coli  K-12 strain is JM108 or a derivative thereof.  
   
   
       7 . The process of  claim 1 , wherein the plasmid has a ColE1-type origin of replication.  
   
   
       8 . The process of  claim 7 , wherein the plasmid is a pUC plasmid.  
   
   
       9 . The process of  claim 1 , wherein said exponential function is  
     
       
         
           
             
               
                 F 
                 t 
               
               = 
               
                 
                   
                     μ 
                     * 
                     
                       X 
                       0 
                     
                   
                   
                     
                       Y 
                       
                         X 
                         / 
                         S 
                       
                     
                     * 
                     
                       C 
                       S 
                     
                   
                 
                 * 
                 
                   ⅇ 
                   
                     μ 
                     ⁢ 
                     
                         
                     
                     ⁢ 
                     t 
                   
                 
               
             
             , 
             wherein 
           
         
       
       F t  is the flow rate [L/h] of the feed medium;  
       X 0  is the total amount of biomass dry cell weight [g] at start of the feeding phase;  
       Y X/S  is the biomass yield coefficient (g dry cell weight per g substrate);  
       C S  is the concentration of said substrate in said feed medium [g/L], and  
       μ is the specific growth rate [h −1 ].  
     
   
   
       10 . The process of  claim 1 , wherein the feeding rate is increased by continuously adding the medium following said exponential function.  
   
   
       11 . The process of  claim 1 , wherein the feeding rate is increased in a semi-continuous mode by adding the medium step-wise following said exponential function.  
   
   
       12 . The process of  claim 1 , wherein the feeding rate is increased in a discontinuous mode by adding the medium pulse-wise following said exponential function.  
   
   
       13 . The process of  claim 1 , wherein the exponential function of the feeding rate is based on measurements of the amount of biomass.  
   
   
       14 . The process of  claim 1 , wherein the growth limiting substrate is a carbon source.  
   
   
       15 . The process of  claim 14 , wherein said carbon source is glucose.  
   
   
       16 . The process of  claim 1 , wherein the growth rate is about. 0.03 to about 0.2 h −1 .  
   
   
       17 . The process of  claim 1 , wherein the medium of the batch culture is comprised of isoleucine.  
   
   
       18 . The process of  claim 17 , wherein the medium of the feeding phase is comprised of isoleucine.  
   
   
       19 . The process of  claim 1 , wherein the medium of the batch culture at the start of fermentation is comprised of ammonium salts as the nitrogen source.  
   
   
       20 . The process of  claim 19 , wherein the ammonium salt is ammonium chloride.  
   
   
       21 . The method of  claim 1 , wherein the pH value of the main culture is adjusted by addition of ammonium hydroxide.  
   
   
       22 . The process of  claim 1 , wherein said culture media are free of antibiotics.  
   
   
       23 . The process of  claim 22 , wherein the culture media used in the pre-culture is free of antibiotics.  
   
   
       24 . A culture medium for producing plasmid DNA in  E. coli  on a manufacturing scale, which is a chemically defined medium that is comprised of 
 a) an organic carbon source selected from glucose, glycerol, fructose, lactose, sucrose, arabinose, or a mixture thereof;    b) an anorganic nitrogen source selected from ammonium salts and ammonium hydroxide, wherein the nitrogen source is present as a component of the medium or added to the medium during fermentation;    c) inorganic salts;    d) optionally one or more substances that complement an auxotrophy of the  E. coli  strain; and    e) isoleucine.    
   
   
       25 . The culture medium of  claim 24 , which is a batch medium present at the start of a batch fermentation or at the start of the batch phase of a fed-batch fermentation and is comprised of: 
 a) glucose in concentration of about 10 to about 30 g/L;    b) an ammonium salt or ammonium hydroxide in a concentration such that the ammonium concentration is about 0.5 to about 2 g/L;    c) inorganic ions that serve as a supply with macro and micro elements;    d) optionally one or more substances that complement an auxotrophy of the  E. coli  strain; and    e) isoleucine in a concentration of about 0.1 to about 0.3 g/L.    
   
   
       26 . The culture medium of  claim 24 , which is a feed medium that is added during the feeding phase of a fed-batch fermentation and is comprised of: 
 a) glucose in concentration of about 300 to about 500 g/L;    a) inorganic ions that serve to supply with macro and micro elements;    b) optionally one or more substances that complement an auxotrophy of the  E. coli  strain; and    c) isoleucine in a concentration of about 6 g/L.